mechanism of multidrug resistance

Biochimica et Biophysics Acta, 948 (1988) 87-128

Elsevier

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BBA 87187

Mechanism of multidrug resistance

Grace Bradley a,b, Peter F. Juranka a and Victor Ling a

n The Ontario Cancer Instiiute, The Princess Margaret Hospital and the Department of Medical Biophysics,

University of Toronto, and b Faculty of Dentistry, University of Toronto, Toronto (Canada)

(Received 17 November 1987)

Contents

I. Introduction . . . . . . . . . . . . . . . . . . . .._................................................

II. The multidrug-resistance phenotype . . . . . . . . . . . . . . . . A. Altered cellular response to drugs . . . . . . . . . . . . . . . B. Transport studies and agents that interfere with drug transport

1. Reduced drug accumulation in multidrug-resistant cells . . . 2. Pharmacologic circumvention of multidrug resistance . . . . .

C. Overexpression of P-glycoprotein . . . . . . . . . . . . . . . . D. Other cellular changes correlated with multidrug resistance

E. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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III. Genetic basis of multidrug resistance ..................................................... A. Cloning of the P-glycoprotein gene ....................................................

1. Monoclonal antibody screening of cDNA expression library for the P-glycoprotein gene ............ 2. Cloning of mRNA overexpressed in multidrug-resistant cell lines by differential hybridization ........ 3. Identification and cloning of DNA sequences amplified in multidrug-resistant cells by in-gel renaturation

B. Expression of P-glycoprotein genes .................................................... 1. P-glycoprotein mRNA overexpression in multidrug-resistant cells ............................ 2. P-glycoprotein mRNA expression in human tissues and tumour samples. ....................... 3. Sequence analysis of P-glycoprotein gene transcripts ......................................

C. Gene amplification in multidrug-resistant cells ............................................ 1. Morphologic evidence ........................................................... 2. Amplification of P-glycoprotein genes ................................................ 3. Differential amplification and evidence for a P-glycoprotein gene family .......................

4. The P-glycoprotein amplicon ...................................................... D. DNA-mediated transfer of multidrug resistance ........................................... E. Summary ......................................................................

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Abbreviations: bp, base pairs; kb, kilobase pairs; kDa, kilodaltons; mRNA, messenger RNA; cDNA, complementary DNA;

SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; HSR, homogeneously staining region; DM, double minute

chromosomes; IMP, intramembrane particles; DHFR, dihydrofolate reductase; [1251]NASV, N-( p-azido[3-‘251]salicyl-N’-(/%

aminoethyl)vindesine; BSO, buthionine sulphoximine; P(BtO),, phorbol 12,13_dibutyrate.

Correspondence: V. Ling, The Ontario Cancer Institute, 500 Sherboume Street, Toronto, Ontario, Canada, M4X lK9.

0304-419X/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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IV. Structure of P-glycoprote in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 A. Sequence analys is of P-glycoprote in - imp l i ca t ions for i ts func t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

B. H o m o l o g y of P-glycoprote in wi th o ther t r anspor t sys tems - evo lu t iona ry cons ide ra t ions . . . . . . . . . . . . . . . . . . . . . 119

V. Mode l for P-glycoprote in funct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

VI. Conc lud ing remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Acknowledgemen t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

I. Introduction

Tumour cell resistance to cytotoxic drugs is considered to be one of the major causes of failure of clinical chemotherapy. Experimental models of drug-resistant tumours have been obtained by selection of cell lines or transplanted turnouts for growth in increasing concentrations of various cytotoxic agents. Characterization of tumour models selected for resistance to colchicine, vincristine, vinblastine, taxol, actinomycin D, daunorubicin and adriamycin has often revealed a multidrug-resistance phenotype in which resistance to the selecting agent is accompanied by cross-resistance to structurally and functionally unrelated cytotoxic agents. Expression of a high-molecular- weight plasma membrane glycoprotein, P-glycoprotein, has been shown to correlate with multidrug resistance in these model systems. Extensive studies have been made of the P-glycoprotein-associated multidrug-resistance phenotype in mouse, hamster and human cells, both in vitro and in vivo. Although studies of other model systems have revealed mechanisms of multidrug resistance that do not involve overexpression of P-glycoprotein, such as alteration of D N A topoisomerase activity, P-glycoprotein-associated multidrug resistance is currently the best characterized in its pharmacologic, biochemical and genetic aspects. Studies of this multidrug-resistance phenotype have provided new insight into various aspects of biology and medicine. For example: (i) mammalian cells can be simultaneously protected against diverse cytotoxic compounds through a pleiotropic membrane alteration; (ii) the transport of many large, amphipathic molecules across the plasma membrane appears to be controlled by a specific mechanism that includes an energy-depen-

dent efflux pathway; (iii) human tumours may display multidrug resistance which is analogous to that characterized in model systems; (iv) multidrug resistance may be amenable to pharmacologic circumvention.

This article reviews the current state of knowl- edge of the P-glycoprotein-associated multidrug- resistance phenotype and its genetic basis and presents the evidence that overexpression of P-glycoprotein causes multidrug resistance. Recent ad- vances in our understanding of the genomic organization of P-glycoprotein genes and the control of their expression are described. The mechanism through which P-glycoprotein mediates the pleiotropic multidrug-resistance phenotype is still incompletely understood. However, a working model for P-glycoprotein structure and function has been formulated from the results of sequence analysis and this provides a conceptual basis for further understanding of the mechanism of P-glycoprotein function in multidrug resistance.

II. The muitidrug resistance phenotype

H-A. A ltered cellular response to drugs

The hallmark of the multidrug-resistance phenotype is cross-resistance to multiple compounds that are unrelated to the selecting agent in structure, cellular target and mode of action. For example, Chinese hamster ovary (CHO) cells selected for resistance to colchicine are cross-resistant not only to the closely related colchicine analogue, colcemid, but also to a wide array of other drugs (Table I). The drugs involved in this phenotype are typically plant alkaloids and antibiotics of bacterial or fungal origin. Their mechanisms of cytotoxic action vary widely and at least one of the drugs (Gramicidin D) is thought to exert its

cytotoxic action at the plasma membrane without entry into cells. Lower levels of cross-resistance to alkylating agents and antimetabolites have been reported in some cases. This phenotype occurs in hamster, mouse and human cell lines selected for resistance to colchicine, vincristine, vinblastine, daunorubicin, adriamycin and actinomycin D [1-10]. In addition, it has been described in trans- plantable tumours selected for drug resistance in vivo [11,12]. It appears that the mechanism of multidrug resistance may be the most common mechanism of resistance against this group of compounds in mammalian cells.

Among clonal multidrug-resistant isolates derived by sequential steps of selection from the same parental cell line, cross-resistance is proportional to resistance against the selecting agent, so that the cross-resistance profiles are similar. How- ever, the cross-resistance patterns that result from selection with different drugs are dissimilar, even if the same cell type is used as the parental cell line (Table I) [3,13]. When a variety of multidrug-resistant cell lines of different origin and selected with different protocols of drug exposure is examined, a considerable degree of variability is noted in the cross-resistance profiles. The highest level of resistance is usually, but not invariably, to the selecting agent. The degree of resistance to structurally similar agents, for example, colchicine and colcemid, or vinblastine and vincristine, is often disparate. Most of the drugs involved in the multidrug-resistance phenotype are amphipathic compounds of high molecular weight. Although early studies of multidrug-resistant cell lines had suggested that the degree of cross-resistance was directly correlated with the molecular weight of the drug [13], subsequent examination of the cross-resistance profiles did not substantiate this hypothesis. Measurement of drug partition coefficients showed some correlation of the degree of cross-resistance with bulk hydrophobicity within a class of compounds [2], but many exceptions to this correlation were noted when a variety of drugs was considered. Thus, while the correlation between resistance to selecting agent and cross- resistance suggests a single underlying mechanism, there is considerable variability in the precise cross-resistance profile so that it cannot be predicted a priori.

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TABLE I

CROSS-RESISTANCE OF MULTIDRUG-RESISTANT CELL LINES

Data for this table were compiled from Refs. 2, 112, 17.

Drug Relative resistance a

CHRC5 b DNRR51 c CEM/ VLB100 a

Colchicine 180 25 45 Colcemid 16 n.a. n.a. Vinblastine 30 22 420 Vincristine n.a. n.a. > 600 Daunorubicin 76 41 120 Adriamycin

(doxorubicin) 25 - 30 110 Gramicidin D = 5000 n.a. n.a. Melphalan 4-15 n.a. n.a. Lidocaine 0.l n.a. n.a. 1-Dehydro-

testosterone 0.1 ~ 1 n.a. Triton X-100 0.3 n.a. n.a.

a Relative resistance was determined by the concentration of drug required to inhibit growth or colony formation in the drug-resistant line compared to the parental line. A value greater than 1 indicates cross-resistance and a value less than 1 indicates collateral sensitivity, n.a. = not analyzed.

b CHRC5 is a clonal CHO cell line selected in three steps for resistance to colchicine [2]. DNRR51 is a clonal CHO cell line selected in two steps for resistance to daunorubicin [112].

d CEM/VLB100 is a human leukaemic cell line selected by continuous culture in increasing vinblastine concentrations [17]. This subline was derived from a clone selected in 100 ng/ml of vinblastine.

Resistance to multiple drugs is associated with increasing sensitivity (collateral sensitivity) to other compounds in some multidrug-resistant cell lines (Table I). Thus, in the CHO cell line (CHRCS) which is 180-fold resistant to colchicine, there is a 2- to 10-fold increase in sensitivity to several local anaesthetics, steroid hormones and some non-ionic detergents. At lower levels of colchicine resistance, the degree of collateral sensitivity is correspond- ingly lower [2]. The mechanism of cytotoxicity of these agents is unknown, although they are all thought to affect plasma membrane function. Re- cently it was shown that a highly multidrug-resistant CHO cell line (CHRB30) is collaterally sensitive to verapamil. The cytotoxic effect of verapamil appears to be exerted at the cell surface because B30 cells are more susceptible than the

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parental cell line AuxB1, despite a greatly reduced uptake of verapamil [14]. Multidrug-resistant mouse SEWA cells were also reported to be collaterally sensitive to verapamil [15]. It should be noted that local anaesthetics, non-ionic detergents and verapamil have dual effects on multidrug-resistant cells, depending on conditions of exposure. When these agents are added in combination with cytotoxic drugs such as vinblastine or adriamycin, they enhance the cytotoxicity of these drugs by interfering with the mechanism of reduced drug accumulation that characterizes multidrug-resistant cells (see subsection II-B). At higher concentrations, these agents alone are cytotoxic, probably by causing membrane damage, and some, but not all, multidrug-resistant cell lines are more susceptible to this latter effect.

H-B. Transport studies and agents that interfere with drug transport

H-B. 1. Reduced drug accumulation in multidrug-resistant cells

With few exceptions, multidrug resistance has been shown to be associated with reduced drug accumulation in different cell lines of hamster, mouse and human origin, which were selected for drug resistance either in vitro [3,13,16-19] or in vivo [20-22]. The time-course of drug accumulation varies with different drugs and among different studies and may be linear or non-linear, but the rate of accumulation is consistently reduced in multidrug-resistant cells when compared to the respective drug-sensitive parent. In those studies where steady state had been reached, the level of drug accumulated in drug-resistant cells has been lower. Furthermore, where sequentially selected cell lines with increasing drug resistance have been compared, drug accumulation has been found to be inversely related to degree of drug resistance [13,16,18].

Studies to determine the basis of the reduced drug accumulation observed in multidrug-resistant cell lines have been undertaken with kinetic measurements using labelled colchicine [16], daunorubicin [20-22], vincristine or vinblastine [17,19,23,24]. In different multidrug-resistant cell lines, increased drug efflux [19,20-24], and/or

decreased drug influx [16,19,23] have frequently been observed. Reduced drug binding or altered intracellular drug distribution [17] have been reported in some systems.

Two points are worth considering in interpre- ting these results. First, analyses of drug transport prove to be technically demanding. For example, influx measurements are complicated by rapid and substantial adsorption of hydrophobic drugs to the cell surface, and correction for such 'instanta- neous' surface binding has been made by diverse techniques in different studies. Moreover, measurements of efflux rates are almost always con- founded by variations in the intracellular 'free drug' concentration. The latter variable depends not only on total cellular drug content, but also on intracellular drug distribution and binding, factors which are incompletely understood for many of the drugs involved. A possible exception is colchicine, whose specific tight binding to cytoplasmic tubulin has been well documented [16]. Second, an altered kinetic property observed with one drug does not necessarily apply to other drugs involved in the multidrug-resistance phenotype, because there are major differences in cellular pharmacokinetics among these drugs. Efflux studies have been primarily undertaken with daunorubicin and vincristine; similar analyses are not feasible for colchicine because of its tight binding to cytoplasmic tubulin [16,25]. Thus, reduced accumulation of colchicine likely reflects only an altered influx process. Conversely, for drugs such as daunorubicin, observations of drug infux as a distinct process are complicated by the influence of drug adsorption, intracellular redistri- bution and drug efflux.

Two different mechanisms, both dependent on metabolic energy, have been proposed to account for the reduced drug accumulation observed in multidrug-resistant cells. (i) Drugs enter cells at nearly normal rates but are removed by an energy-dependent efflux pump that operates with a greater capacity in drug-resistant cells [20,22,23]. (ii) An energy-dependent permeability barrier controls drug entry into cells, and operates with greater efficiency in drug-resistant cells [13,25]. Data in support of these models are summarized below. Such divergence in the mechanisms proposed to account for multidrug resistance may reflect the

variation in experimental approach, where different data have been gathered using different drugs.

Reduced accumulation of the anthracycline antibiotics and vinca alkaloids is thought to result from the enhanced activity of an energy-dependent efflux pump [20-24]. Energy deprivation (by incubation with inhibitors of oxidative phosphorylation in a glucose-deficient medium) resulted in increased steady-state drug levels in both drug- sensitive and drug-resistant cells, the effect being greater in the latter, so that the difference between drug-sensitive and resistant cells was greatly diminished. Addition of glucose induced rapid drug release from the cells, the release being greater in resistant cells, so that the difference in intracellular drug levels between drug-sensitive and resistant cells was re-established. In Chinese hamster lung cells and in P388 leukaemia cells, reduced accumulation and resistance to these drugs was reversed by the non-ionic detergent Tween 80 [26,27]. Thus it appears that the activity of the energy-dependent drug efflux pump can be inhibited by non-ionic detergents.

In contrast to the apparent large difference in energy-dependent efflux between drug-sensitive and resistant cells, the measured influx of these drugs appeared to be relatively unaltered in multidrug-resistant cells. Different studies have attributed influx to either passive diffusion or mediated transport [19-21,23]. Trans-inhibition studies using vincristine showed that, for both drug-sensitive and drug-resistant Chinese hamster lung cells, efflux of preloaded, unlabelled drug inhibited influx of labeled drug, the effect being greater in drug-resistant cells. This suggests that influx and efflux pathways interact and are not completely independent [19].

As noted above, reduced accumulation of colchicine in CHO cells is thought to be directly related to decreased influx. In studies of colchicine transport in both drug-sensitive and resistant cells, drug uptake remained linear over the first 2 h. Energy deprivation resulted in increased rates of colchicine uptake, the increase being greater in resistant cells. Glucose addition caused an im- mediate reduction of colchicine uptake rate to values characteristics of each cell line prior to energy deprivation. The kinetics of the stimulation of drug uptake upon energy depletion and the

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subsequent reversal upon glucose addition paral- leled the fluctuation in ATP levels, suggesting that the effects of energy deprivation were mediated through changes in ATP levels [28,29]. These results led to the hypothesis of an energy-dependent permeability barrier to colchicine that operates with greater efficiency in drug-resistant cells to reduce colchicine accumulation.

Local anaesthetics and non-ionic detergents were found to increase colchicine accumulation in both drug-sensitive and resistant cells. Under conditions of maximal stimulation, drug-resistant cells attained uptake rates similar to the drug-sensitive parent. Local anaesthetics and non-ionic detergents are known to protect red blood cells against hypotonic lysis, probably by interacting with the erythrocyte membrane. Local anaesthetics have also been shown to interact with membrane lipids and thus increase membrane fluidity and alter permeability. These observations are consistent with the hypothesis that altered membrane permeability is important in reduced colchicine accumulation [30].

Colchicine uptake was shown to be affected by other drugs to which cross-resistance is observed in colchicine-resistant CHO cells. It was surprising to find that colchicine uptake was increased, rather than decreased, by increasing concentrations of vinblastine, vincristine, daunorubicin and actinomycin D. The concentration of vinblastine required for a a given level of stimulation of colchicine uptake was proportional to the degree of drug resistance. When colchicine uptake was maximally stimulated by the presence of appropriate concentrations of vinblastine, the variation in colchicine accumulation among sublines of different degrees of resistance was essentially abolished [30], These findings suggest that drugs such as vinblas- fine and daunorubicin may have properties similar to non-ionic detergents and local anaesthetics.

The determinants of cellular accumulation of the different drugs involved in the multidrug-resistance phenotype appear to be complex and variable according to the drug being studied. Consider- ation of the experimental data on drug transport suggests that the two proposed mechanisms of drug efflux pump and drug permeability barrier are not mutually exclusive but may represent different aspects of a pleiotropic alteration in mem-

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brane function that results in reduced accumulation of a diverse group of drugs.

H-B.2. Pharmacologic circumvention of multidrug resistance

A diverse group of compounds has been found to reverse the reduction in drug accumulation in multidrug-resistant cells and thus circumvent multidrug resistance. Studies of these compounds have been directed towards two objectives: (i) consideration of their known effects on cell physiology may help elucidate the mechanism of reduced drug accumulation; (ii) modifying agents with lit- tle or no cytotoxic effects may be useful for administration in conjunction with cytotoxic drugs to overcome multidrug resistance in clinical chemotherapy.

The effect of metabolic inhibitors and membrane-active agents such as local anaesthetics and non-ionic detergents on drug accumulation has been described above. Numerous other compounds also interfere with the mechanism of multidrug resistance. They can be divided into the following groups: (i) non-cytotoxic analogues of anthracyclines and vinca alkaloids; (ii) calcium channel blockers and calmodulin inhibitors; (iii) other compounds that do not clearly belong to the above groups.

A non-cytotoxic structural analogue of daunorubicin, N-acetyldaunorubicin, has been found to increase daunorubicin accumulation in Ehrlich ascites tumour cells by inhibition of active daunorubicin efflux. The increase in daunorubicin uptake was greater in drug-resistant cells than drug- sensitive cells, so that simultaneous administration of N-acetyldaunorubicin and daunorubicin counteracted resistance of these cells to daunorubicin, both in vitro and in vivo [31].

In a more extensive study of non-toxic drug analogs, the cytotoxic effect of daunorubicin, adriamycin, vincristine and vinblastine in multidrug-resistant P388 cells (P388/VCR and P388/ADR) was potentiated by simultaneous administration of structural analogues. These analogues had no cytotoxic effects of their own at the concentrations used, but appeared to enhance cytotoxicity by inhibiting effiux of the cytotoxic drugs and thus increasing cellular accumulation of these drugs. It is interesting to note that the

vinblastine analogues vindoline, was able to inhibit active efflux of daunorubicin in P388/VCR cells and that, in general, structural analogs of vinca alkaloids that counteracted resistance to vincristine were also effective in counteracting resistance to daunorubicin through increase in drug accumulation [32,33]. This suggests that the active drug efflux mechanism which is characteristically enhanced in multidrug-resistant cells is shared by at least two groups of structurally dissimilar drugs, the anthracycline antibiotics and the vinca alkaloids.

The calcium channel blocker, verapamil, has been found to counteract vinca alkaloid resistance in P388 leukaemia cells both in vitro and in vivo [34]. Non-cytotoxic doses of verapamil increased cytotoxicity of vincristine and vinblastine in P388 cells and their multidrug-resistant derivative P388/VCR, through enhanced accumulation and retention of the cytotoxic drug. The effect of verapamil was more marked in drug-resistant cells so that the difference in drug response between sensitive and resistant cells was completely abolished in vitro. Furthermore, the resistance to vincristine treatment in mice bearing P388/VCR leukaemia was partly circumvented by co-administration of verapamil. Simultaneous presence of verapamil and the cytotoxic drug was required for potentiation of drug effect and there was no evidence of damage to membrane integrity by verapamil, indicating that the effect of verapamil on the mechanism of multidrug resistance was reversible and specific.

It was subsequently shown that calcium antagonists other than verapamil, as well as calmodulin inhibitors such as trifluoperazine, can increase accumulation and retention of vincristine, adriamycin and daunorubicin in drug-resistant P388 sublines, resulting in partial or complete reversal of drug resistance [35,36,37]. A similar effect was obtained with reserpine, a membrane- active agent that affects calcium content of nerve and muscle cells [38].

Reversal of drug resistance by calcium channel blockers and calmodulin inhibitors has now been demonstrated for other multidrug-resistant mouse, hamster and human cell lines selected for drug resistance in vivo [39] and in vitro [14,17,18,40-43]. Partial or complete reversal of resistance to drugs

such as vincristine, vinblastine, adriamycin, daunorubicin and colchicine has been demonstrated. In studies where drug transport was measured in the presence of these 'chemosensitizers', increased drug accumulation and retention were found to accompany potentiation of drug effect. In addition, it was recently reported that similar concentrations of verapamil (1-2 /xM) could reverse chloroquin resistance in strains of the malarial parasite Plasmodium faciparum which exhibit cross-resistance to multiple antimalarial agents [44]. Chloroquin resistance in P. falciparum is associated with decreased drug accumulation as a result of enhanced release of drug. Verapamil appears to overcome chloroquin resistance by inhibiting drug release and increasing drug accumulation, in a manner very similar to reversal of multidrug resistance in mammalian cells [45].

Calcium channel blockers and calmodulin inhibitors have distinct effects on cellular physiology that are still incompletely understood, but both classes of compounds affect intracellular calcium levels. Accordingly, it was speculated that efflux of cytotoxic drugs might be controlled by calcium-calmodulin complex or other calcium-dependent processes [34,35]. Experiments in which 45Ca uptake was measured failed to demonstrate differences in calcium flux between drug-sensitive and multidrug-resistant cells or any effect of calcium antagonists on calcium flux in these cells [14,37]. Ability to circumvent multidrug resistance did not correlate well with effect on calcium chan- nels or calmodulin [35,43,46]. These results should not be considered to have excluded the involvement of calcium in drug transport processes, but nevertheless, they have led to consideration of calcium-independent mechanisms for circumvention of drug resistance. Both calcium channel blockers and phenothiazine calmodulin inhibitors have lipophilic portions that are compatible with membrane binding, so these compounds may alter drug uptake and retention through membrane effects. Recently, verapamil was shown to bind specifically to isolated membrane vesicles of multidrug-resistant cells and to inhibit vinblastine- analog photoaffinity labelling of P-glycoprotein in membrane vesicles (see Section V) [43]. The relationship between these findings and the increase in drug accumulation and retention caused by

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verapamil in intact cells is not clear, but it appears that verapamil itself may be a substrate of the energy-dependent drug efflux pathway. In a number of instances, multidrug-resistant cells accu- mulate and retain less verapamil than the drug- sensitive parent, in a pattern that is similar to that seen for drugs such as vinblastine and daunorubicin [14,47].

The search for agents to circumvent multidrug resistance has led to various compounds that are not known to be calcium channel blockers or calmodulin inhibitors, including quinidine [43,48], chloroquin [49], triparanol derivatives such as tamoxifen [50], propanolol [51], synthetic isopre- noids [52] and cyclosporin A [139]. These compounds were studied in different multidrug-resistant cell lines, where they were shown to overcome resistance to a number of cytotoxic agents, including vinblastine, vincristine, daunorubicin, adriamycin, colchicine and actinomycin D. In general, the enhancement of cytotoxic effect was related to increased drug accumulation and retention. Many of these substances are amphipathic in nature and some are known to be membrane-active. A recent study showed a correlation between reversal of multidrug resistance and inhibition of vinblastine-analogue photoaffinity labelling of P- glycoprotein in membrane vesicles [140] (see Sec- tion V). Thus multidrug resistance may be reversed through interference with drug binding by P-glycoprotein. It was pointed out that calcium channel blockers, calmodulin inhibitors and other compounds such as those listed above might interfere with drug accumulation and retention by different mechanisms, so that extrapolation of mechanism from one compound to another might be unjustified [37,47].

The study of circumvention of multidrug resistance by a variety of compounds has strengthened the association between multidrug resistance and reduced drug accumulation and retention. Re- cently, a novel analysis of doxorubicin (adriamycin) cytotoxicity in two multidrug-resistant cell lines in the presence of graded concentrations of several calcium channel blockers was reported. It was shown that resistance to adriamycin in both multidrug-resistant cell lines could be explained quantitatively by reduced drug accumulation [53]. The exact mechanism(s) by which these 'chem-

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osensitizers' increase drug accumulation and retention in multidrug-resistant cells is not known, partly because so many compounds with ap- parently different effects on cell physiology are able to interfere with this phenotype. Neverthe- less, the finding that compounds such as verapamil and trifluoperazine can circumvent multidrug resistance in tumour models has stimulated considerable interest in the development of chemotherapeutic treatment protocols in which verapamil or similar agents may be combined with appropriate cytotoxic drugs to overcome clinical drug resistance.

H-C Overexpression of P-glycoprotein

Biochemical studies of multidrug-resistant cells have been focused on the plasma membrane because the altered responses to diverse compounds described above are evidence for changes of plasma membrane structure and function. The most consistent finding has been the overexpression of a high-molecular-weight glycoprotein, P-glycoprotein. Initially, increased amounts of a 170 kDa cell surface glycoprotein were demonstrated in colchicine-selected multidrug-resistant CHO cells, relative to the drug-sensitive parent, by using the techniques of reductive tritiation of surface glycoproteins and metabolic labelling with [14C]glucos- amine. Since the resistance to multiple drugs that characterizes these colchicine-selected CHO mutants has been shown to be the consequence of reduced accumulation, it was postulated that the overexpressed 170 kDa membrane glycoprotein, termed P-glycoprotein, modulated plasma membrane properties to reduce drug accumulation [54]. Partial purification of P-glycoprotein from colchicine-resistant CHO cells was achieved through differential centrifugation of cell homogenates and lectin column affinity chromatography, confirm- ing that P-glycoprotein is an integral membrane glycoprotein of 170 kDa overexpressed in colchicine-selected multidrug-resistant CHO cells. The level of expression of P-glycoprotein was shown to correlate with degree of drug resistance and the amount of P-glycoprotein was greatly diminished in drug-sensitive revertants [55].

Preparation of antiserum against plasma membrane vesicles of the colchicine-resistant CHO cell

line CHRC5 and adoption of an immunoblotting technique in conjunction with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) improved the specificity and sensitivity of detection of P-glycoprotein. Two studies using the immunoblot technique to identify P-glycoprotein established the significance of P-glycoprotein in the multidrug-resistance phenotype. (i) It was shown that multidrug resistance could be transferred through DNA transfection from hamster CHRC5 cells to drug-sensitive mouse LTA cells. In all independent transformants analyzed, acquisition of multidrug resistance was associated with overexpression of P-glycoprotein [56]. (ii) Multidrug-resistant CHO sublines obtained by selection with daunorubicin also overexpressed a 170 kDa surface glycoprotein which displayed immunological cross-reactivity with P-glycoprotein identified in colchicine-selected CHO cells [3]. Ex- amination of a number of multidrug-resistant cell lines of hamster, mouse and human origin demonstrated in every case that multidrug resistance correlates with increased expression of a 170 kDa surface antigen which cross-reacts immunologi- cally with P-glycoprotein of CHRC5 origin [57]. Thus, P-glycoprotein is conserved in size and immunological reactivity across species, and consistently associated with multidrug resistance. Its level of expression is proportional to degree of drug resistance. Low levels of P-glycoprotein can just be detected in the drug-sensitive parental cell lines. This suggests that P-glycoprotein is a plasma membrane constituent of drug-sensitive cells and that its overexpression plays a central role in multidrug resistance.

Development of monoclonal antibodies directed against three spatially distinct epitopes of P-glycoprotein further increased the specificity of its detection [58]. A survey of a large series of independently derived multidrug-resistant cell lines by immunoblot analysis using a monoclonal antibody against P-glycoprotein (C219) supported the earlier findings of the close correlation between overexpression of P-glycoprotein and multidrug resistance and the conserved nature of P-glycoprotein [59]. Detection of P-glycoprotein in plasma membrane preparations requires attention to technical considerations, as P-glycoprotein appears to be relatively refractory to certain staining

and solubilization procedures. Furthermore, the behaviour of P-glycoprotein in gel electrophoresis has been found to be significantly affected by variables such as method of isolation of plasma membranes and type of gel electrophoresis used [59] (Greenberger, L., personal communication). Thus, the high-molecular-weight cell-surface glycoproteins that were independently described and characterized in various multidrug-resistant cell lines - gp150-180 in Chinese hamster lung cells [13], gp170-190 in human leukaemic cells [60], P180 in Chinese hamster lung cells [61] and the 130-150 kDa phosphoglycoproteins in mouse macrophage-like cells [6,62] - have been shown to co-migrate with a C219-reactive protein component in the appropriate gel electrophoresis system, indicating their identity with P-glycoprotein [59] (Center, M., personal communication; Horwitz, S., personal communication).

The characteristic diversity of the cross-resistance pattern of different multidrug-resistant cell lines is an important consideration in attempts to understand the mechanism through which P-glycoprotein may mediate multidrug resistance. A small but consistent difference was noted in the apparent molecular weights of P-glycoprotein derived from colchicine-selected CHO sublines and that derived from daunorubicin-selected CHO sublines [3]. Analysis of gp 150-180 (P-glycoprotein) from various multidrug-resistant hamster DC-3F sublines by two-dimensional gel electrophoresis showed some heterogeneity in size and charge that was suggestive of the existence of a family of related molecules [1]. The high-molecular-weight phosphoglycoproteins that were described in three multidrug-resistant mouse J774.2 derivatives, selected with colchicine, vinblastine or taxol, respectively, were found to be heterogeneous in size (varying between 130 and 150 kDa), although they shared immunological cross-reactivity as shown by immunoblot analysis. Char- acterization of cyanogen bromide-cleaved peptides of these homologous, multidrug resistance-associated phosphoglycoproteins by immunoblotting further illustrated that they were similar but not identical. These membrane glycoproteins have been shown to be completely analogous to P-glycoprotein as characterized earlier and they represent the most distinct illustration of the variability

95

of P-glycoprotein molecules overexpressed in multidrug-resistant derivatives selected with different drugs and displaying distinct cross-resistance profiles [6,62].

The basis of heterogeneity of P-glycoprotein expressed by different multidrug-resistant J774.2 derivatives was investigated by studying the biosynthesis of P-glycoprotein in pulse-chase experiments and by analysis of N-linked oligosaccharides on both precursor and mature forms of P-glycoprotein in these cell lines. Differences in N-linked oligosaccharides could account for the variation in electrophoretic mobility of P-glycoprotein that was overexpressed in colchicine- selected and vinblastine-selected J774.2 derivatives. In contrast, the difference in electrophoretic mobility between the two antigenically related forms of P-glycoprotein found to be overexpressed in taxol-selected J774.2 cells could not be attributed to variation of N-linked oligosaccharides. This heterogeneity was demonstrated in both precursor and mature forms of the protein and might be due to distinct polypeptide backbones or post- translational modifications other than N-linked glycosylation [141]. Further work is required to completely elucidate the mechanism(s) that gener- ate heterogeneous forms of P-glycoprotein and to determine whether this is the biochemical basis for diversity of cross-resistance patterns.

The conservation of P-glycoprotein structure across mammalian species and its detection at low levels in drug-sensitive cell lines indicate that P- glycoprotein may be an important membrane component of mammalian cells. The occurrence of P-glycoprotein in normal tissues has recently been investigated. In a study of P-glycoprotein expression in non-neoplastic human tissues using an immunoblot assay with a monoclonal antibody against P-glycoprotein (C219), a variety of intraabdominal organs were studied and plasma membrane samples from liver consistently showed relatively high levels of P-glycoprotein. Membrane samples from small bowel displayed lower levels of P-glycoprotein than those from liver and in the remaining intraabdominal organs that were tested; P-glycoprotein could not be detected [63]. It is possible that low levels of expression of P-glycoprotein in the tested tissues other than liver and small bowel were below the limits of detection by

96

the immunoblot assay used. Moreover, since solid tissue samples were used for membrane preparations, the immunoblot assay would only reflect an average of many cell types, including connective tissue cells. This might have effectively obscured relatively high levels of P-glycoprotein expression in certain cell types.

Recently, the expression of P-glycoprotein in normal human tissues was studied at a cellular level with immunohistochemical techniques, using a monoclonal antibody (MRK16) against human P-glycoprotein. Among 17 different tissues that were examined as frozen sections, 6 showed high levels of P-glycoprotein in a specific distribution. These tissues were liver, pancreas, kidney, colon, jejunum and adrenal. With the exception of the adrenals, P-glycoprotein was always localized to the luminal or apical surface of cells - on the surfaces that line bile canaliculi, bile ductules, pancreatic ductules, proximal renal tubules and the jejunum and colon. These findings indicate that P-glycoprotein is expressed at relatively high levels in a small number of cell types within non-neoplastic tissues and suggest that P-glycoprotein may have a physiologic role in secretory processes [142]. Studies of P-glycoprotein expression in normal tissues through measurement of mRNA content will be described in subsection III-B.2.

Human tumour samples of various histologic types have also been analyzed for P-glycoprotein. The consistent correlation between increased expression of P-glycoprotein and multidrug resistance in experimental systems suggests that P-glycoprotein overexpression may be the basis of cellular drug resistance in clinical turnouts, particularly with respect to drugs shown to be involved in the multidrug-resistance phenotype, such as vincristine, adriamycin and actinomycin D. Detec- tion of P-glycoprotein overexpression in tumour samples may facilitate diagnosis of tumour cell resistance to multiple chemotherapeutic agents so that alternative or novel chemotherapy protocols could be used to circumvent resistance and im- prove treatment results. In preliminary studies using either Western blots or immunocytochemi- cal staining, levels of P-glycoprotein comparable to those found in multidrug-resistant human cell lines were demonstrated in some cases of ovarian

carcinoma, leukaemia and sarcoma of different histologic subtypes [64-66]. Further studies are required to optimize the detection of P-glycoprotein in tumour samples and to establish the relationship between level of P-glycoprotein expression and clinical outcome of chemotherapy. Nev- ertheless, the demonstration of elevated P-glycoprotein levels in some tumour samples clearly indicates that P-glycoprotein overexpression is not limited to experimental tumour models.

H-D. Other cellular changes correlated with multidrug resistance

While the association of P-glycoprotein with multidrug resistance is well documented, a wide array of other biochemical changes have also been identified to distinguish multidrug-resistant cells from their drug-sensitive parent (Table II). In general, these changes have been found only in a subset of multidrug-resistant cell lines which overexpress P-glycoprotein. Some of these alterations have been studied in serially derived multidrug-resistant cell lines and their revertants, and have been found to correlate with the degree of drug resistance.

A small, acidic cytosolic protein (19-22 kDa) variously termed sorcin/V19, CP22, p21, 22 kDa polypeptide or 21 kDa protein, has been described in numerous multidrug-resistant cell lines in a number of independent investigations [8,67-70]. Sorcin/V19 was initially identified as a small, acidic (20 kDa, pI 5.7) cytosolic protein overexpressed in several vincristine-selected multidrug- resistant cell lines. Preparation of antiserum against sorcin facilitated the detection of this protein in other multidrug-resistant cell lines by immunoblotting and it was determined that sorcin is conserved in size and immunological reactivity across species. It is present in a variety of multidrug-resistant cell lines of mouse, hamster and human origin selected with vincristine, actinomycin D, colchicine and adriamycin, but is absent from a number of other multidrug-resistant cell lines. The antisorcin antiserum cross-reacts with an independently identified 22 kDa acidic (pI 5.3) cytosolic protein called CP2: that is overexpressed in the multidrug-resistant hamster cell line CHRC5

and an adriamycin-selected mouse cell line EMT6- AR1. Both sorcin and CP22 have been found to bind calcium in an in vitro calcium-binding assay and it appears that they represent the same protein [67,68]. In cells that overexpress sorcin/CP22, this cytosolic protein represents a major calcium- binding protein, and this may affect calcium metabolism in these multidrug-resistant cells. The relationship between sorcin/CP22 and the other small cytoplasmic proteins is less clear, but their similarity in size and charge as illustrated by two- dimensional gel electrophoresis and their common association with multidrug resistance suggest that they may be homologous proteins.

The basis for the close correspondence between P-glycoprotein overexpression and sorcin overexpression in multidrug-resistant cells has been elucidated through molecular genetic studies (see subsection Ill-CA). The sorcin gene was found to be closely juxtaposed to the P-glycoprotein gene in both hamster and human genomes and overexpression of the sorcin gene appears to be the result of its co-amplification with the P-glycoprotein gene. The inconsistent occurrence of sorcin gene amplification and overexpression in multidrug-resistant cells suggests that sorcin overproduction may not be necessary for the development of multidrug resistance. Nevertheless, the overexpression of sorcin in numerous independently isolated multidrug-resistant cell lines indicates that sorcin may be an important modulator of the multidrug- resistance phenotype.

Among the changes in enzyme activity that have been described, the alteration in glutathione transferase activity is of particular interest for two reasons: (i) glutathione transferase, particularly the isozyme that was shown to be elevated, can protect cells against free-radical damage, thought to be one of the modes of cytotoxic action of anthracyclines such as adriamycin. Thus it has been proposed that the enhanced glutathione transferase activity constitutes part of the mechanism of resistance against adriamycin and possibly other drugs involved in the multidrug-resistance phenotype; (ii) the same isozyme of glutathione transferase is found at elevated levels in carcinogen-induced hyperplastic rat liver nodules, suggesting that there may be parallel cellular responses during chemical carcinogenesis and devel-

97

opment of multidrug resistance [71,72]. In act inomycin D and vincristine selected multidrug-resistant mouse SEWA cells, where increased glutathione transferase activity was also detected, there was no clear correlation between the increase in enzyme activity and level of drug resistance [15]. The contribution of this.enzyme activity to multidrug resistance remains to be established.

The role of glutathione in multidrug resistance has also been addressed by measurement of glutathione levels in drug-sensitive and resistant cells and also by attempting to overcome drug resistance with buthionine sulphoximine (BSO), which inhibits the rate-limiting step in glutathione synthesis and thus depletes cellular glutathione levels. The glutathione level of adriamycin-resistant human ovarian carcinoma cells (A2780 A°) was found to be slightly higher than that of the parental A2780 cells. Exposure to minimally toxic concentration of BSO resulted in partial depletion of glutathione (70-90%) in both drug-sensitive and drug-resistant cells. This was associated with some increase in susceptibility to adriamycin, the effect being greater in the drug-sensitive parent and relatively small in the drug-resistant variant. It appeared that partial reduction in glutathione levels could not overcome adriamycin resistance in this system to a significant degree [73]. Similar studies performed on P388 cells and their multidrug-resistant derivatives P388/ADR showed no correlation between adriamycin resistance and glutathione levels. Furthermore, there was no enhancement of daunorubicin cytotoxicity in either drug-sensitive or resistant cells after reduction of glutathione levels by approx. 90% with BSO treatment [74].

Certain biological and physical changes at the cellular level have been described as accompany- ing multidrug resistance. The best known biological change is the "reverse transformation" phe- nomenon observed in multidrug-resistant hamster and mouse cells [13,67], where multidrug-resistant cells have altered in vitro growth characteristics and decreased tumorigenicity. Physical attributes of multidrug-resistant cells that have been documented include an increase in susceptibility to mechanical disruption [55], both an increase and decrease in plasma membrane fluidity [75-78] and

TA

BL

E I

I

BIO

CH

EM

ICA

L C

HA

NG

ES

IN

MU

LT

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UG

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SIS

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LL

LIN

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revi

atio

ns:

AC

T-D

, ac

tino

myc

in D

; A

DR

, ad

riam

ycin

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CH

, col

chic

ine;

CH

L,

Chi

nese

ham

ster

lun

g; C

HO

, C

hine

se h

amst

er o

vary

; D

NR

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unor

ubic

in;

VC

R,

vinc

rist

ine;

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B,

vinb

last

ine.

Bio

chem

ical

alt

erat

ion

Occ

urre

nce

Com

men

ts

Ref

s.

A. P

rote

in/p

oly

pep

tid

e In

crea

se in

170

kD

a pl

asm

a m

embr

ane

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o-

prot

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(P-g

lyco

prot

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Incr

ease

in

150-

180

kDa

mem

bran

e gl

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pr

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p150

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)

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170-

190

kDa

mem

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pr

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Incr

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in 1

80 k

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mem

bran

e pr

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180)

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in

13

0-15

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prot

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Dec

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pro-

te

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m

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mem

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Dec

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roup

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chem

ical

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ease

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of

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67

68

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icat

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., p

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ST

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ITY

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LT

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UG

-RE

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TA

NT

CE

LL

S

Abb

revi

atio

ns:

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1L a

dria

myc

in;

FP

, fl

uore

scen

ce p

olar

izat

ion;

ES

R,

elec

tron

spi

n re

sona

nce

spec

tros

copy

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l li

ne

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hniq

ue

Alt

erat

ion

Com

men

ts

Ref

s.

in m

embr

ane

flui

dity

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se M

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of

who

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ells

in

crea

se

in

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easi

ngly

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ug-r

esis

tant

su

blin

es,

redu

ctio

n in

dr

ug

75

K2

subl

ines

ac

cum

ulat

ion

is c

orre

late

d di

rect

ly w

ith

incr

ease

in

mem

bra

ne

flui

dity

ES

R

incr

ease

ad

riam

ycin

re

sist

ance

co

rrel

ated

di

rect

ly

wit

h in

crea

se

in

76

mem

bran

e fl

uidi

ty,

thou

ght

to r

elat

e to

cel

l su

rfac

e cy

toto

xlci

ty

of a

dria

myc

in r

athe

r th

an c

hang

es i

n dr

ug a

ccum

ulat

ion

FP

of

plas

ma

mem

- in

crea

se

loss

of

resi

stan

ce in

rev

erta

nts

is n

ot

acco

mpa

nied

by

dec

reas

e 77

br

ahe

prep

arat

ions

in

mem

bran

e fl

uidi

ty

ES

R a

nd F

P

decr

ease

de

crea

se i

n m

embr

ane

flui

dity

at

trib

uted

to

al

tere

d ph

os-

78,

80

phol

ipid

com

posi

tion

and

tho

ught

to

resu

lt i

n re

duce

d dr

ug

accu

mul

atio

n

AD

R

sele

cted

m

ouse

S

180

sarc

oma

cells

AD

R s

elec

ted

Chi

nese

ham

ster

lu

ng c

ells

AD

R

sele

cted

P

38

8

mou

se

leuk

aem

ia c

eils

an increase in the rate of plasma membrane endocytosis [79].

Several studies of plasma membrane lipid structural order or 'membrane fluidity' have been made by electron spin resonance spectroscopy or fluorescence polarization studies of appropriately labelled whole cells or plasma membrane preparations (Table III). It can be seen that the relationship between membrane fluidity and anthracycline resistance is not consistent among different re- ports and that there are different interpretations of the basis of the association between membrane fluidity and anthracycline cytotoxicity. The finding of increased membrane fluidity in some studies of drug-resistant cells prompted a study of membrane endocytosis in anthracycline-resistant Ehrlich ascites tumour cells, since the rate of this process had previously been shown to be affected by changes in membrane fluidity [79]. Using cationized ferritin as a plasma membrane marker, it was shown that drug-resistant cells had a higher rate of endocytosis than the drug-sensitive parent. Since the total plasma membrane area remained constant during the experiment, and since the rate of plasma membrane biosynthesis was much lower than the rate of endocytosis, the accelerated endocytosis probably reflected an increase in cycling between the plasma membrane and the endosomal-lysosomal system.

These biological and physical changes may be the cellular manifestation of one or more of the biochemical changes described earlier. For example, one could speculate that overexpression of a large, integral plasma membrane glycoprotein such as P-glycoprotein may affect susceptibility of the plasma membrane to disruption or cause differences in the disposition of membrane probes used in membrane fluidity measurements. The decrease in membrane fluidity found by one group of investigators was indeed attributed to changes in membrane lipid composition [80]. In general, however, the precise biochemical explanation for these cellular changes that sometimes accompany multidrug resistance is not known, nor is their contribution to the mechanism of multidrug resistance understood. Indirect evidence has been cited to support the hypothesis that the increase in membrane endocytosis that was demonstrated in anthracycline-resistant Ehrlich tumour cells (see

101

above) constitutes part of the mechanism for enhanced drug efflux. For example, membrane endocytosis is rapid (occurring in minutes) and energy-dependent; agents such as chloroquin that interfere with endosomal-lysosomal function affect drug accumulation and retention in multidrug-resistant cells [79]. It seems that further investigations into alterations in cellular behaviour that accompany multidrug resistance would com- plement biochemical and molecular genetic studies in the overall effort to understand the mechanism of multidrug resistance.

H-E. Summary

The multidrug-resistance phenotype is characterized by simultaneous alterations in cellular response to many compounds of different struc- tures, including cytotoxic agents useful in chemotherapy. The precise effects of most of these compounds on cells are not clear and this has hampered attempts to understand the basis of this phenotype. The major site of action of cytotoxic agents such as vincristine, actinomycin D and colchicine resides within cells and currently available evidence suggests that resistance is due to lowered drug concentration at the respective intracellular targets of such drugs. Alterations in drug accumulation involving influx, efflux, as well as changes in intracellular compartmentalization may all effect a decrease in drug concentration at the critical target sites and evidence has been presented for the occurrence of all of these changes. In addition, some of the compounds affected by the multidrug-resistance phenotype, such as Gra- micidin D, local anaesthetics and non-ionic detergents and possibly verapamil, disturb the plasma membrane when present in appropriate concentrations, and altered responses to such compounds signify a change in plasma membrane properties. The pleiotropy of the multidrug-resistance phenotype does not in itself constitute an argument for involvement of multiple mechanisms, as has often been proposed. Alteration in function of a complex organelle such as the plasma membrane could result in a pleiotropic effect as described above.

At the biochemical level, overexpression of the conserved plasma membrane glycoprotein, P-glycoprotein, has been shown to be the most con-

102

sistent alteration in multidrug-resistant cells. Evi- dence has been presented to support the hypothesis that P-glycoprotein is a normal plasma membrane constituent and its overexpression plays a central role in the development of multidrug resistance. Various biochemical changes in addition to P-glycoprotein overexpression have been reported in some multidrug-resistant cell lines. The significance of these biochemical changes in multidrug resistance is not clear at present. They may represent ancillary mechanisms that modulate the multidrug-resistance phenotype in certain cell types. The occurrence of some ot these changes may be linked to P-glycoprotein overexpression at the genetic level (see subsection III- C.4), or at the level of shared cellular biochemical pathways. Such linkages may be expected to vary among different cell types or among cells selected for multidrug resistance in independent experiments.

An important feature of the multidrug-resistance phenotype is the variability of the cross-resistance profiles among different multidrug-resistant cell lines. Collateral sensitivity to local anaesthetics and non-ionic detergents also appears to be a variable feature. Independent investigations have demonstrated small variations in the apparent molecular weight and charge of P-glycoprotein from different multidrug-resistant cell lines, suggesting that a family of P-glycoprotein molecules may exist. Differential expression of the P-glycoprotein family of proteins may partly account for the variability of the multidrug-resistance phenotype. In addition, the cellular environ- ment in which P-glycoprotein overexpression occurs would be expected to vary among multidrug- resistant cell lines of different origin. Thus, the interaction between overexpressed P-glycoprotein molecules and other cellular constituents that are present at wild-type levels may also account for some of the variability of the phenotype.

The multidrug-resistance phenotype is un- doubtedly complex, but its attributes are consistent with one major underlying mechanism. In- tensive study of multidrug resistance at the genetic level has provided evidence to support this hypothesis and has also provided molecular probes to begin to unravel the complexity of this phe- nomenon.

III. Genetic basis of multidrug resistance

III-A. Cloning of the P-glycoprotein gene

The complexity of the multidrug-resistance phenotype naturally raises the question of whether multiple genetic events are required. Early studies of colchicine-selected multidrug-resistant CHO cells have provided evidence that alteration in a single gene or a small number of closely linked genes forms the basis of the entire phenotype of resistance to the selecting drug, cross-resistance to other unrelated drugs, collateral sensitivity to certain membrane-active agents and overexpression of plasma membrane P-glycoprotein. Evidence in support of this conclusion includes: (i) independent clones selected for colchicine resistance in a single step, with or without mutagenic treatment, all display the complete multidrug-resistance phenotype; (ii) revertants isolated in a single step without mutagenic treatment show concomitant loss of multidrug resistance and P-glycoprotein overexpression; (iii) multidrug resistance and increase in P-glycoprotein are similarly expressed in a co-dominant fashion in somatic cell hybrids; and (iv) independent clones of DNA transformants obtained by transfer of DNA from multidrug-resistant cells to drug-sensitive cells all show the complete pleiotropic phenotype. Since unlin- ked genes are not likely to be co-transferred in many independent transformants, this argues strongly that a single genetic event is sufficient for the multidrug-resistance phenotype [81].

The availability of monoclonal antibodies directed against independent epitopes of P-glycoprotein has allowed sensitive and specific detection of P-glycoprotein in immunoblots. Thus it has been demonstrated that P-glycoprotein is overexpressed in a variety of rodent and human multidrug-resistant cell lines in proportion to the degree of multidrug resistance, and that this represents the most consistent biochemical change in multidrug-resistant cells (see above, subsections II-C and II-D).

Taken together, these observations strongly suggest that an alteration in the P-glycoprotein gene is the basis of the multidrug-resistance phenotype. Low levels of expression of P-glycoprotein in drug-sensitive parental cells and progressive

increase in multidrug resistance and P-glycopro- rein expression after multiple steps of selection are most consistent with a mechanism of P-glycoprotein gene amplification, similar to gene amplification associated with DHFR enzyme overproduction in methotrexate resistance [58,81]. Within the past few years, intensive efforts have been directed at the study of the molecular genetics of the multidrug-resistance phenotype. Based on the considerations discussed above, a direct approach

103

would be to clone the P-glycoprotein gene using the available monoclonal antibodies [82,83]. Alter- natively, a number of investigators have cloned genes that are commonly amplified and overexpressed in various multidrug-resistant cells [84-88]. Characterization of several independently cloned genes that are consistently amplified and overexpressed in different multidrug-resistant cell lines reveals that they all encode P-glycoprotein (Table IV). Thus, P-glycoprotein gene amplification and

TABLE IV

PUTATIVE P-GLYCOPROTEIN DNA CLONES

Method of isolation Resultant DNA Size of mRNA detected Refs. clones on Northern blot

Monoclonal antibody screening of colchicine-resistant Chinese pCHP1 4.7 kb 82 hamster ovary cell cDNA expression library

Monoclonal antibody screening of colchicine-resistant Chinese 3.6 kb-P170 4.5 kb hamster ovary cell cDNA expression library (minor 2.2 kb) 83

Isolation of amplified genomic DNA from adriamycin-re- pDRI.1 none 86 sistant Chinese hamster lung cells as detected by in-gel DNA renaturation

Screening of adriamycin-resistant Chinese hamster lung cell 87 genomic library using pDRI.1

Screening of vincristine-resistant Chinese hamster ovary cell 93 genomic library using pDRI.1

Screening of drug-sensitive mouse pre-B cell cDNA library 94 using pDR7.8 and pDR1.6

Screening of colchicine-resistant human KB/HeLa cell ge- 88 nomic library using pDR4.7

Screening of colchicine-resistant human KB/HeLa cell cDNA library using pHDR4,4

Screening for human genomic sequences with the Alu-repeat ~KA2.6 DNA probe in a library created from a mouse cell transformed with DNA from an adriamycin-resistant human leukaemia cell line

Screening of colchicine-resistant Chinese hamster ovary cell cDNA library for overexpressed transcripts using single-strand cDNA from sensitive and resistant cells

Screening of vincristine-resistant Chinese hamster lung cell p5L-18 cDNA library for overexpressed transcripts using Cot 10-300 genomic DNA from sensitive and resistant cells

Screening of adriamycin-resistant human breast cancer cell pADR1 cDNA library using p5L-18

pDR7.8, pDR1.6, pDR4.7

pDR6, pDR7

XDRll , hDR29

pHDR4.4 (subclone pMDR1) pHDR4.5 (subclone pMDR2)

hHDR10, hHDR5, hHDR104

cp20, cp28 (class 2 clones)

5 kb (minor 2 kb)

4.3 kb

5 kb

4.5 kb

none

4.5 kb (minor 10.5 kb)

4.5kb

4.5 kb

4.5 kb (minor 2.3 kb)

4.8 kb

98 100

110

84

85

126

104

overexpression appear to be the predominant genetic alteration in multidrug-resistant cells.

III-A.1. Monoclonal antibody screening of cDNA expression library for the P-glycoprotein gene

A monoclonal antibody against a highly conserved epitope of P-glycoprotein (C219, see Ref. 58) was used as a probe to isolate cDNA clones that encode P-glycoprotein from a ~gt l l expression library prepared from a highly multidrug-resistant CHO cell line (CHRB30) [82]. A single clone with a 660 bp insert was obtained and several lines of evidence were used to demonstrate that this cloned cDNA fragment (pCHP1) represented a portion of the P-glycoprotein gene: (i) the polypeptide encoded by pCHP1 was shown by immunoblot analysis to be recognized by monoclonal antibodies that identify three independent epitopes of P-glycoprotein; this demonstrated un- equivocally that the pCHP1 polypeptide was part of P-glycoprotein; (ii) Northern blot analysis showed pCHP1 to detect a 4.7 kb mRNA which was overexpressed in CHRB30 relative to the drug-sensitive parental cell line, AuxB1. The size of the mRNA was consistent with the estimated molecular weight of the polypeptide portion of P-glycoprotein, while slot blot analysis demonstrated increasing amounts of this mRNA in a series of increasingly drug-resistant CHO cell lines selected with colchicine; (iii) Southern blot analysis using pCHP1 under stringent hybridization conditions revealed DNA sequences that were amplified in multidrug-resistant hamster, mouse and human cell lines. In the series of colchicine-selected CHO cell lines with increasing multidrug resistance, drug resistance correlated directly with plasma membrane P-glycoprotein content and with level of pCHPl-homologous mRNA and DNA [82]; (iv) sequence analysis of additional cDNA clones isolated using pCHP1 revealed a protein structure consistent with that of an integral membrane protein [89]. Thus it was concluded that the cDNA clone, pCHP1, encodes a portion of P-glycoprotein.

Recently, other investigators used similar meth- odology to clone a cDNA fragment from a ~gt l l expression library prepared from the multidrug-resistant CHO cell line CHRC5 by screening with another monoclonal antibody against P-glycopro-

tein [83]. This 3.6 kb cDNA fragment most probably represents a portion of the P-glycoprotein gene, since it detects a 4.5 kb mRNA that is overexpressed in the CHRC5 cell line relative to the parent, AuxB1.

III-A.2. Cloning of mRNA overexpressed in multidrug-resistant cell lines by differential hybridization

The technique of differential hybridization was used by two groups of investigators to isolate cDNA clones corresponding to genes that are overexpressed in multidrug-resistant cells compared to the parent cells [84,85]. It was reasoned that such genes should include those that controlled multidrug resistance. In one study [84] a cDNA library prepared from the multidrug-resistant CHO cell line CHRC5 was screened by sequential hybridization to labelled single-strand cDNA from CHRC5 (drug-resistant) and from AuxB1 (drug-sensitive) cells. Colonies which pref- erentially hybridized to the probe from drug-resistant ceils were analyzed. Six classes of cDNA clone were distinguished on the basis of their hybridization to RNA transcripts of different sizes on Northern blots [84,90]. The corresponding genomic sequences for all six classes were found to be amplified in CHRC5 cells relative to AuxB1. Thus, at least six genes are amplified and overexpressed in the multidrug-resistant cell line CH R C5. The class 2 cDNA clones recognize a 4.5 kb mRNA that is overproduced in CHRC5 cells. Representative class 2 cDNA clones cross-hybridized to the P-glycoprotein cDNA clone, pCHP1 (described above) and subsequent sequence analysis of class 2 clones confirmed that they encode portions of P-glycoprotein (Endicott, J., Juranka, P. and Ling, V., unpublished observations). Further study of the amplification and overexpression of these six genes in three other independently derived multidrug-resistant hamster cell lines showed that the P-glycoprotein gene is the only one consistently amplified and overexpressed, while the other genes appear to be co- amplified in some of the cell lines owing to their location contiguous to the P-glycoprotein gene [91,92] (for further discussion, see subsection III- C.4 on the P-glycoprotein amplicon).

In another, independent study of overexpressed genes in multidrug-resistant cells, a cDNA library

from a highly multidrug-resistant hamster cell line (DC-3F/VCRd-5L) was screened by sequential hybridization to Cot-fractionated genomic DNA from DC-3F/VCRd-5L ceils and from the drug-sensitive parent, DC-3F [85]. Cytogenetic studies had indicated that multidrug resistance in this cell line was associated with gene amplification, manifested cytologically as the presence of homogeneously staining regions (HSR). Accord- ingly, Cot fractions of DNA were chosen to enrich the probe for amplified sequences. A single cDNA clone (p5L-18) was selected on the basis of preferential hybridization to amplified DNA from drug-resistant cells. Sequences homologous to p5L-18 were found to be amplified and overexpressed in other independently derived multidrug- resistant cell lines of hamster, mouse and human origin, indicating that this sequence was highly conserved and closely correlated with multidrug resistance. The p5L-18 insert recognized a major mRNA species of 4.5 kb and a minor species of 2.3 kb on Northern blots and cross-hybridized with class 2 cDNA clones derived from CHRC5 cells which were known to encode P-glycoprotein (see above). Hence, it was deduced that p5L-18 was a cDNA clone for P-glycoprotein.

111-.4.3. Identification and cloning of DNA sequences amplified in multidrug-resistant cells by in- gel renaturation

The technique of in-gel DNA renaturation was used to detect DNA sequences commonly amplified in two independently derived, multidrug-resistant hamster cell lines, LZ and CHRC5, but which were not amplified in the respective drug- sensitive parents [86]. One of these commonly amplified fragments was cloned (pDRI.1). Al- though the degree of amplification of pDRI.1 was correlated with the degree of drug resistance in both hamster cell lines, its expression could not be detected on Northern blots. Subsequently, pDRI.1 was used as the initial hybridization probe to isolate a series of overlapping genomic clones from the multidrug-resistant cell line LZ. These clones defined a contiguous domain of genomic DNA of approx. 120 kb that was shown to be amplified in both LZ and CHRC5 ceils. Northern blot analysis indicated that this DNA domain contains a gene (called the multidrug-resistance gene, mdr) that

105

encodes a 5 kb mRNA which is overexpressed in both LZ and CHRC5 cells in proportion to their degree of multidrug resistance [87].

The cloned fragment pDRI.1 was also used to isolate overlapping genomic DNA clones from a cosmid library of another multidrug-resistant Chinese hamster cell line (CHO/VCRS). These clones represented a DNA domain of about 45 kb that was amplified in CHO/VCRS. DNA sequences from within this cloned segment recognized a 4.3 kb mRNA that was overexpressed in this multidrug-resistant cell line. Several lines of evidence, including the similarity in size of the transcript detected on Northern blot, suggested that the cloned DNA segment was related to the P-glycoprotein gene [93].

Clones from the 120 kb hamster genomic DNA domain (the mdr sequences) contain sequences which are conserved among rodent and human cells. This proved to be useful for the isolation of homologous sequences in mouse and human cells. Thus, two cDNA clones (~DRl l and hDR29) representing the mouse homologues of mdr were isolated from a drug-sensitive mouse cell line. The clone h D R l l was shown by transfection assay to confer multidrug resistance on drug-sensitive recipient cells [94]. Moreover, the multidrug-resistant transfectants overexpress P-glycoprotein, as shown by immunoblots probed with the P-glycoprotein monoclonal antibody, C219 (Gros, P., personal communication). Sequence analysis of XDR11, which encompasses an entire protein coding region, revealed a protein structure consistent with an integral membrane glycoprotein [95]. Comparison of the amino-acid sequence derived from XDRll and that derived from P-glycoprotein cDNA clones showed extensive sequence homology [96]. Thus it can be concluded that XDRll, which corresponds to the mouse mdr gene, encodes P-glycoprotein.

A DNA probe derived from the hamster mdr domain (pDR4.7) was also used to identify homologous DNA sequences in a human carcinoma cell line (KB-HeLa) and its multidrug-resistant derivatives. Two genomic DNA clones, pHDR4.4 and pHDR4.5, were isolated by screening a genomic library of a colchicine-selected multidrug- resistant KB subline (KB-C3) with the pDR4.7 hamster probe. These were distinct sequences, as

106

shown by restriction pattern analysis, but they shared sequence similarities in the region which hybridized to the pDR4.7 probe and thus cross- hybridized with each other under conditions of low stringency. Southern blot analysis of multidrug-resistant KB sublines selected with three different drugs (colchicine, vinblastine or adriamycin), using DNA probes derived from pHDR4.4 and pHDR4.5 (pMDR1 and pMDR2 probes, respectively) revealed that sequences homologous to pMDR1 and pMDR2 were differentially amplified in multidrug-resistant cell lines, pMDR1 sequences were amplified in all the multidrug-resistant KB sublines and were also shown to be overexpressed as a 4.5-5 kb mRNA in Northern blot analysis of these sublines. In contrast, pMDR2 sequences were only amplified in some multidrug- resistant KB sublines and their overexpression could not be detected in any of the multidrug-resistant sublines. These findings suggested that pMDR1 and pMDR2 might be part of two related but distinct genes, probably different members of a multigene family, and accordingly, these genes were designated mdrl and mdr2 [88]. The pMDR1 probe (for mdrl sequences) was used to isolate a series of overlapping cDNA clones from the multidrug-resistant KB subline, KB C2.5. These clones were shown by sequence analysis to constitute a complete mdrl gene transcript [97,98]. The amino-acid sequence of mdrl shares extensive homology with both the mouse mdr sequence and the hamster P-glycoprotein sequence [96]. In addition, mdrl cDNA clones cross-hybridize with P-glycoprotein cDNA clones [99].

It can be seen that several independent efforts to clone genes associated with multidrug resistance, using different multidrug-resistant cell lines of hamster, mouse or human origin have led to the isolation of DNA sequences highly homologous to hamster P-glycoprotein sequences, as demonstrated by direct sequence comparison or by hybridization analysis. Further evidence that these cloned sequences encode all or part of P-glycoprotein is recapitulated as follows: (i) the cellular source of all initial DNA or cDNA clones is highly multidrug-resistant cell lines known to overexpress P-glycoprotein at high levels (CHRC5, DC-3F/VCRd-5L, LZ); (ii) in Northern blots the cloned DNA probes recognize an mRNA of ap-

prox. 4.5 kb that is overexpressed in multidrug-resistant cell lines but not detected in drug-sensitive cells. In many, although not all, of the multidrug- resistant cell lines examined, these probes hybridize to DNA sequences that are amplified relative to the drug-sensitive parent. Since these multidrug-resistant cell lines have been shown by immunoblot analysis to overexpress P-glycoprotein in proportion to their degree of drug resistance, the mRNA overexpression and DNA amplification as detected by these cloned sequences correspond to P-glycoprotein overexpression; (iii) transfection of the mouse ~DRl l cDNA clone into drug-sensitive hamster cells results in overproduction of P-glycoprotein by the recipient cells.

It is remarkable that the diverse approaches taken to isolate genes associated with multidrug resistance have all led to the P-glycoprotein gene. Molecular genetic analysis of multidrug-resistant cell fines thus corroborates earlier biochemical analysis which demonstrates that P-glycoprotein is highly conserved in structure across species and that its overexpression is the most consistent biochemical alteration in multidrug-resistant cells. Furthermore, the availability of independently derived, cloned probes for P-glycoprotein has allowed extensive studies of the genomic organization of P-glycoprotein and the genetic control of P-glycoprotein expression in cell lines and tissues.

III-B. Expression of P-glycoprotein genes

III-B.1. P-glycoprotein mRNA overexpression in multidrug-resistant cells

The P-glycoprotein gene probes described in the section above have been used to analyze a variety of multidrug-resistant cell lines for expression of P-glycoprotein at the mRNA level, using either Northern blots or RNA slot blots. In all cell lines examined to date, development of multidrug resistance is associated with increased amounts of P-glycoprotein mRNA [82,83,85,91,100]. In these studies using different multidrug-resistant cell lines, the size of P-glycoprotein mRNA has been estimated from Northern blots to be 4.5-5 kb. Minor RNA transcripts of 2-2.3 kb and of 10.5 kb have also been reported in some studies [83,85,87,98]. For many of these cell lines, P-glycoprotein overexpression has also been demon-

strated at the protein level by immunoblot analysis using monoclonal antibodies against P-glycoprotein. It appears that elevated levels of P-glycoprotein mRNA in these cell lines are translated into increased amounts of the protein. Among cell lines that were serially derived by selection for growth in increasing concentrations of drugs, step- wise increases could be demonstrated in drug resistance, plasma membrane P-glycoprotein content and P-glycoprotein mRNA levels [82,85,99].

In the clonal series of colchicine-selected CHO cells derived from AuxB1, increase in P-glycoprotein mRNA and protein is associated with progressive P-glycoprotein gene amplification in every step of selection [82]. In contrast, during development of increasing degree of multidrug resistance in a series of colchicine-selected human KB/HeLa sublines, increase in P-glycoprotein mRNA pre- cedes P-glycoprotein gene amplification, that is, acquisition of low levels of multidrug resistance involves only increased expression of P-glycoprotein genes without increase in gene copy number [100]. Similarly, among multidrug-resistant derivatives of the human ovarian carcinoma cell line SKOV3, selected with either vinblastine or vincristine, early steps in the development of multidrug resistance are associated with increase in P-glycoprotein at the protein and mRNA levels only. Subsequent steps in selection result in sublines with P-glycoprotein gene amplification and further increases in degree of multidrug resistance and P-glycoprotein mRNA and protein content (Bradley, G., Naik, M. and Ling, V., unpublished data).

There are other examples where multidrug resistance is accompanied by increased P-glycoprotein expression in the absence of gene amplification. Multidrug-resistant P388 leukaemia sublines developed by selection with adriamycin, vincristine or actinomycin D in vivo were shown to overexpress P-glycoprotein in immunoblots probed with monoclonal antibody C219, but only the adriamycin-selected subline had amplified P-glycoprotein genes (Gerlach, J. and Ling, V., unpublished data). Recently, an adriamycin-selected multidrug-resistant breast cancer cell line (MDA- 231) was also reported to overexpress P-glycoprotein mRNA, as shown by Northern blot analysis, without concomitant amplification of P-glycopro-

107

tein genes [83]. The regulatory mechanisms that are involved in overexpression of P-glycoprotein genes in the absence of gene amplification are presently unknown.

III-B.2. P-glycoprotein mRNA expression in human tissues and tumour samples

Analyses of human tissues and tumour samples for P-glycoprotein have been carried out using immunoblot assays (see subsection II-C). Similar analyses have been performed at the genetic level using P-glycoprotein gene probes. A preliminary survey of a variety of human tissues and tumors for P-glycoprotein mRNA using the mdrl/P-glycoprotein gene probe in Northern blot and slot blot analyses has been reported [101]. The results suggest that there are significant differences among tissues in the level of expression of P-glycoprotein mRNA, the highest levels being in kidney and adrenal gland, intermediate levels in lung, liver, colon and rectum and low levels in other tested tissues. The number of samples of each tissue that was analyzed was generally small and there appeared to be considerable variability of P-glycoprotein mRNA levels among samples of the same tissue. It is possible that variability of P-glycoprotein expression exists among individuals and this may confound any interpretation of tissue-specific variation of expression unless complete surveys of major tissues of a sufficient number of individuals are undertaken. Subsequent study of the expression of P-glycoprotein in normal human tissues using immunohistochemical staining revealed a generally similar distribution of P-glycoprotein among tissues and demonstrated a specific cellular localization of the protein within tissues that showed a high level of P-glycoprotein mRNA [142] (see subsection II-C).

The levels of P-glycoprotein mRNA in normal hamster tissues were measured in a sensitive RNAase protection assay, using a probe derived from a hamster P-glycoprotein cDNA clone. The pattern of expression of hamster P-glycoprotein mRNA found in this study was distinctly different from that reported for human tissues. In the hamster, the highest level of P-glycoprotein mRNA was found in oesophagus, testis and uterus, while adrenal gland, kidney and liver showed low levels of P-glycoprotein mRNA. It appeared that sub-

108

stantial differences might exist between species in the tissue-specific pattern of P-glycoprotein expression [143].

Overexpression of P-glycoprotein mRNA was demonstrated in carcinogen-induced, preneoplastic and neoplastic rat liver nodules [144,145]. An established protocol for induction of rat hepatomas was used which included administration of two carcinogens, diethylnitrosamine (DEN) and 2- acetylaminofluorene (2-AAF), and partial hepatectomy. In one of these studies, the level of expression of P-glycoprotein mRNA in preneoplastic and neoplastic liver nodules was comparable to that seen in a multidrug-resistant cell line showing approx. 100-fold increase in resistance to drugs [144]. The elevation in P-glycoprotein expression appeared to be stable over a period of months, because similar levels of P-glycoprotein mRNA were found in preneoplastic nodules removed 6-8 weeks after initiation of chemical carcinogenesis as in hepatocellular carcinomas removed 6-8 months after initiation. The role of P-glycoprotein overexpression in chemical carcinogenesis is presently not clear, especially since partial application of the carcinogenic treatment protocol (2-acetylaminofluorene administration with hepatectomy, but without diethylnitrosamine treatment) did not result in preneoplastic nodules but did result in marked elevation of P-glycoprotein mRNA levels [145]. Treatment of rats with 2-acetylaminofluorene in conjunction with hepatectomy may provide a useful in vivo model system to study the regulation of P-glycoprotein expression without involving prolonged selection for drug resistance as in conventional models of P-glycoprotein overexpression. In addition, studies of P-glycoprotein overexpression in carcinogen-treated tissues may help in the understanding of primary resistance to chemotherapeutic agents in malignant diseases thought to be associated with environmental carcinogens, such as colon carcinoma.

Elevated levels of P-glycoprotein mRNA were detected in some tumour samples of a variety of histologic types, the RNA levels being comparable to those of moderately multidrug-resistant human KB carcinoma cell lines [101]. Some of these samples were from tumours previously treated with combination chemotherapy. As in the case of

analysis of tumour samples for P-glycoprotein using immunoblot assays (subsection II-C), a sys- tematic evaluation of the correlation between level of P-glycoprotein expression and clinical response to chemotherapy has not been feasible, but these RNA studies were in general agreement with the protein studies, that a fraction of clinical tumour samples exhibited elevated levels of P-glycoprotein and that in some cases, this was associated with poor response to chemotherapy.

III-B.3. Sequence analysis of P-glycoprotein gene transcripts

Northern blot analysis of RNA from drug-sensitive cell lines has revealed low levels of P-glycoprotein mRNA in some studies [84]. However, in studies of other drug-sensitive cell lines, P-glycoprotein mRNA has not been detectable. It seems likely that P-glycoprotein is expressed at low levels in drug-sensitive cells, levels which may be below the usual limits of detection in typical Northern blots. A more fruitful approach has been to isolate and sequence cDNA clones that correspond to P-glycoprotein transcripts from drug sensitive- cells. Several findings of fundamental importance emerged from these studies: (i) P-glycoprotein is encoded by a family of genes; in three independent studies of different drug-sensitive cell lines and tissues of hamster, mouse and human origin, at least two gene family members were found to be expressed in each case; (ii) differences among P-glycoprotein gene family members appear to be conserved across species - thus, the multigene family was established prior to divergence of mammalian species; (iii) a P-glycoprotein cDNA clone derived from a drug-sensitive cell line could confer the multidrug-resistance phenotype when transfected and overexpressed in a drug-sensitive recipient. This implies that P-glycoprotein that is expressed in drug-sensitive cells, albeit at lower levels, does not differ fundamentally in function from that expressed in multidrug-resistant cells (also see subsection III-D).

The P-glycoprotein cDNA probe, pCHP1, was used to isolate a number of partial cDNA clones from a drug-sensitive Chinese hamster cell line (E29 Pro + ). Comparative sequence analysis indicates that these clones correspond to transcripts from two distinct P-glycoprotein gene family

members, designated pgpl and pgp2. Further- more, each P-glycoprotein gene has two alternate polyadenylation sites, so that at least four P-giycoprotein transcripts are simultaneously expressed in this drug-sensitive cell line [96]. A third P-glycoprotein gene family member (pgp3) was subsequently identified through sequence analysis of a genomic clone, P13, isolated from a drug-sensitive hamster cell by its hybridization to pCHP1 (Ng, W. and Ling, V., unpublished data). However, additional rounds of screening of the drug-sensitive cell line cDNA library and characterization of a total of 24 cDNA clones by sequence analysis revealed only clones derived from pgpl or pgp2 transcripts, suggesting that pgp3 might not be expressed in this cell line.

Two distinct but related cDNA clones ( h D R l l and XDR29) have been isolated from a drug-sensitive mouse pre-B cell line by their cross-hybridization to hamster mdr/P-glycoprotein sequences (see subsection III-A.3). XDRll is a virtually complete cDNA clone and has been shown by transfection assay to confer the multidrug-resistance phenotype when overexpressed in drug-sensitive recipient cells [94,102]. Sequence comparison between XDRll and the hamster P-giycoprotein cDNA clones indicates that h D R l l is the mouse homologue of the hamster pgp2 gene [96]. Preliminary sequence comparison between ?~DR29 and the hamster P-glycoprotein gene family indicates that XDR29 corresponds to the hamster pgp3 gene (Gros, P., personal communication).

Recently, a number of P-glycoprotein cDNA clones were isolated from human liver cDNA libraries through cross-hybridization with a class 2 (hamster P-giycoprotein) cDNA clone (see subsection III-A.2). Sequence analysis reveals that some of the clones represent transcripts from the human mdrl gene that has previously been identified and described in multidrug-resistant human KB/HeLa sublines (see subsection III-A.3) and found to be homologous to the hamster pgpl gene by sequence comparison [96]. The remaining P-glycoprotein cDNA clones isolated from the human liver libraries represent transcripts from a second human P-glycoprotein gene and preliminary sequence analysis supports its assignment as the human homologue of hamster pgp3 [103].

The finding that the P-glycoprotein gene family

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structure is conserved across species suggests that differential expression of gene family members may be important in modulating P-glycoprotein function. It appears from the sequence analysis of cDNA clones described above that drug-sensitive cell lines or tissues simultaneously express at least two of the three P-glycoprotein genes, possibly in a tissue-dependent manner. The pattern of expression of P-giycoprotein genes in cell lines selected for multidrug resistance has not been analyzed to a similar extent. Northern blot analysis of multidrug-resistant cell lines (see subsection III-B.1) has not revealed differences in P-glycoprotein transcripts that can be correlated with variations in cross-resistance profile. One important consideration is that a P-glycoprotein gene probe such as pCHP1 that contains sequences highly conserved among gene family members would be unable to distinguish among transcripts of similar size from different gene family members. It should now be possible to develop probes specific for pgpl, 2 and 3 that will be useful in the qualitative and quantitative evaluation of expression of the P-glycoprotein gene family in various multidrug-resistant cell lines. A converse consideration is that attempts to isolate P-glycoprotein sequences may result in cloning of P-glycoprotein gene segments that are not conserved among gene family members; such cloned sequences, when used solely as probes for P-glycoprotein expression, may fail to detect expression of some of the P-glycoprotein genes. Thus, further analysis of the structural and functional relationship among P-glycoprotein gene family members is essential for progress in understanding how P-glycoprotein over-expression leads to the complex multidrug-resistance phenotype.

III-C. Gene amplification in multidrug-resistant cells

III-C.1. Morphologic evidence Cytogenetic studies reveal that homogeneously

staining regions (HSR) and double minute chromosomes (DM) are frequently present in multidrug-resistant cell lines (Table V). In some cases, the number of DM or the length of HSR correlates with degree of multidrug resistance. Cells selected for increasing drug resistance display increased DM and HSR, while loss of these cyto- logic markers is evident in cells which have re-

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TABLE V

CYTOLOGICAL EVIDENCE FOR GENE AMPLIFICATION IN MULTIDRUG-RESISTANT CELL LINES

Abbreviations: DM, double minute chromosome; HSR, homogeneously staining region; ABR, abnormal band region.

Cell line/selecting drug Cytological

evidence

of gene

amplification

Comments Refs.

Chinese hamster ovary

Cytochalasin D HSR HSR on chromosome 1; HSR absent in revertants


vincristine: VCR15 DM amplified pDR2-4 (P-glycoprotein) sequences localized

VCR5 and VCR15 ABR to lq and Zlq by in situ hybridization

Chinese hamster lung (DC-3F)

Vincristine: VCRd HSR length of HSR proportional to degree of drug resistance

VCRm ABR

Actinomycin D: ADX absent P-glycoprotein genes shown to be amplified in VCR,

Daunorubicin: DMXX absent ADX and DMXX lines [85,91]

Chinese hamster lung

Adriamycin DM number of DM proportional to degree of drug resistance

Mouse neuroblastoma

Maytansine: MT-2 DM unstable drug-resistant phenotype in absence of drug

loss of DM in drug-sensitive revertant

Maytansine: MT-3 absent stable phenotype

Mouse transformants

Colchicine DM correlation between levels of resistance, number of DM

and level of P-glycoprotein as detected by immunoblot

assay

Mouse tumour cell line (MAZ)

Vincristine DM


colchicine: CHRB30

colchicine: CH R C5

DM, HSR

HSR

amplified P-glycoprotein sequences localized to HSR

on 24 by in situ hybridization

amplified P-glycoprotein and contiguous linked genes

localized to HSRs on 7q and 27; wild type P-glycoprotein

genes localized to Iq26

number of DM proportional to degree of drug resistance;

P-glycoprotein genes shown to be amplified [85]

82

92

127

93

128

129

130

131

128

Mouse tumour cell line (SEWA)

Actinomycin D

Colchicine

Vincristine

Actinomycin D

HSR,‘DM

DM

DM

DM loss of DM in revertants

number of DM proportional to level of resistance

132

Mouse macrophage-like cells (5774.2)

Tax01 DM

Colchicine DM

DM absent in revertants 6

number of DM proportional to degree of drug resistance 7

Djungarian hamster

Colchicine (low)

Colchicine (high)

Adriablastin (low)

DM

HSR/DM

absent

additional chromosome 4 HSR present on chromosome 4; loss of DM or HSR in

revertants correlates with level of resistance

additional chromosome 4

134

TABLE V (continued)

111

Cell line/selecting drug Cytological of gene evidence amplification

Comments Refs.

Adriablastine (medium) DM Adriablastin (high) HSR

Human carcinoma (KB/HeLa) colchicine DM Adriamycin DM Vinblastine DM

Human neuroblastoma (SH-SY5Y) vincristine DM

Human myeloma (RPMI-8226) Doxorubicin absent

Human breast cancer (MCF-7) Adriamycin HSR

DM absent in revertant 135

P-glycoprotein genes shown to be amplified [85] 128

7q anomaly present; wild-type P-glycoprotein gene 9 localized to 7q21-36 in human cells [136,137,126]

amplified P-glycoprotein sequences localized to HSR 126 by in situ hybridization

verted to a more drug-sensitive phenotype. These chromosome aberrations are indicative of gene amplification in mammalian cells [104]. Thus, the cytogenetic findings provided one of the early indications that the genetic basis for the multidrug-resistance phenotype might reside in gene amplification and overexpression.

III-C.2. Amplification of P-glycoprotein genes

Southern blot analysis of numerous multidrug- resistant cell lines of hamster, mouse and human origin, using various cloned P-glycoprotein gene probes (see subsection III-A) have shown that development of multidrug resistance is often, but not always, associated with P-glycoprotein gene amplification [82,85,87,91,100]. Among cell lines selected for increasing level of multidrug resistance, the level of gene amplification generally correlates with degree of drug resistance [82,85]. The finding that, in some cases, early steps in the development of multidrug resistance can occur through overexpression of P-glycoprotein without gene amplification has been discussed earlier. Fur- ther evidence to suggest that expression of P-gly-

coprotein may be controlled at the mRNA level, independent from control of gene copy number is provided by studies where semiquantitative measurements of P-glycoprotein DNA and RNA levels showed disproportionate overexpression of P-glycoprotein genes in some multidrug-resistant cell lines [85,91]. However, interpretation of these estimates of P-glycoprotein DNA and RNA levels was complicated by the existence of multiple P- glycoprotein genes. Since the genes were not sep- arately analyzed in the Southern and Northern blot techniques that were employed, lack of transcription of some of the gene family members could account for part of the discrepancy between DNA and RNA levels.

In situ hybridization of P-glycoprotein gene probes to chromosome spreads has localized amplified P-glycoprotein sequences to HSRs that were previously described in some multidrug-resistant cell lines (Table V) [82,92]. Conversely, many multidrug-resistant cell lines now known to have amplified P-glycoprotein sequences do not have identifiable HSR or DM, probably because the total length of amplified sequences is insuffi- cient to produce a visible abnormality in

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metaphase spreads. Thus the presence or absence of cytogenetic abnormalities in multidrug-resistant cell lines is not an indication of qualitative differences in the genetic basis of drug resistance.

Ili-C.3. Differential amplification and evidence for a P-glycoprotein gene family

Southern blot analysis of genomic DNA from hamster, mouse and human cells using the P- glycoprotein cDNA probe pCHP1 reveals multiple DNA fragments. The large number of bands detected by this relatively small (660 bp) probe is consistent with the existence of a family of P- glycoprotein genes (see subsection III-B.3) most or all of which contain segments recognized by pCHP1. Within the clonal series of multidrug-resistant CHO cell lines derived from AuxB1 by colchicine selection, progressive amplification of pCHPl-homologous bands is seen in every step of selection. The simultaneous amplification of all bands in'CHRA3, which was isolated in a single step from the drug-sensitive parent, indicates that the gene family members are probably clustered within one amplicon. In the more highly drug-resistant cell lines CHRC5 and CHRB30, two subsets of pCHPl-homologous bands are amplified to

different levels, suggesting that gene family members may be differentially amplified during selection for multidrug resistance. Similarly, in the multidrug-resistant mouse ECH R and human CEM-VLB cell lines, development of multidrug resistance is associated with amplification of only a subset of the DNA segments recognized by pCHP1 [82].

Four independently derived, multidrug-resistant hamster cell lines have been compared with respect to P-glycoprotein gene amplification using a class 2 (P-glycoprotein) cDNA probe. The five DNA fragments that are detected by this probe may be divided into three subsets on the basis of their differential amplification (Fig. 1). Two multidrug-resistant derivatives of the DC-3F cell line obtained independently by selection with vincristine (DC-3F/VCRd-5L) or actinomycin D (DC- 3F /ADX) , as well as the CHRC5 cell line, display the same pattern of differential amplification of DNA fragments, where the five fragments are divided into two subsets. In view of the finding of identical patterns of amplification among three independently derived cell lines, the most likely explanation of differential amplification is that subsets of DNA fragments that are amplified to a

30x

.... f 1 } r, ¢', ~,HR 5 [ I I 4x 6x

D C - 3 F / A D X ~3Ox I J

6x 8x D C - 3 F / D M X X , n

15Ox

I I i I

13ox I

4Ox 130x

D C - 3 F / V C R d - 5 L . . . . , [ I g e n e c lass 1 2 a 2 b 2c 3 4 5

Fig. 1. Tentative map of genes amplified in multidrug resistance. The gene order is based on pulsed-field gradient gel analyses of the CHRC5 genome and the amplification levels of the individual genes (represented by blocks) in the four resistant cell lines. The amplification levels are indicated above the blocks. Gene class 2 represents the three P-glycoprotein gene family members and gene class 4 has been identified as sorcin/V19. A sixth gene class, amplified in CHRC5, has been found and it is located adjacent to class 4 and 5, though the exact order of these genes is not yet known. This figure has been taken from de Bruijn et al. [91] with permission.

different extent correspond to distinct P-glycoprotein gene family members. In another multidrug- resistant DC-3F derivative (DC-3F/DMXX), selected with daunorubicin, one of the two subsets of DNA fragments can be further subdivided on the basis of two observed levels of amplification. Thus Southern blot analysis of these hamster cell lines corroborates the finding from sequence analysis of P-glycoprotein clones that there are at least three homologous but distinct P-glycoprotein genes [911.

An independent study of P-glycoprotein gene amplification in multidrug-resistant DC-3F derivatives using the p5L-18 cDNA probe (see subsection III-A.2) also showed differential amplification of P-glycoprotein sequences [85]. The DNA fragments that were detected differed from those reported in the previous study [91] because the two P-glycoprotein probes hybridized to different but overlapping segments of the P-glycoprotein gene. It is clear from these two studies of the DC-3F sublines that the pattern of amplification cannot be predicted according to the drug used for selection or the cross-resistance profile. Thus, two DC-3F sublines that were isolated independently using vincristine as the drug of selection in each case displayed a different pattern of amplification of P-glycoprotein sequences, while the same pattern of amplification was observed for a group of DC-3F sublines selected with three different drugs [85]. One explanation for this lack of correspondence may be that the pattern of DNA amplification detected by a single P-glycoprotein probe only offers an incomplete representation of the differential amplification of P-glycoprotein genes in a multidrug-resistant cell line. Analysis of Southern blots using probes that specifically recognize each gene family member should provide more precise information on differential amplification of P-glycoprotein genes and its effect on the multidrug-resistance phenotype.

Cloned fragments of genomic P-glycoprotein sequences (the hamster mdr gene probes and the human mdrl probe, see subsection III-A.3) have also been used for Southern blot analysis of multidrug-resistant cell lines and the respective drug-sensitive parental lines. In these studies, only one or two bands were recognized in each cell line and they were shown to be amplified in propor-

113

tion to the degree of multidrug resistance [87,100]. These P-glycoprotein sequences detected by genomic DNA probes appear to correspond to a single member of the P-glycoprotein gene family. The hamster mdr probes hybridized weakly to a second set of DNA fragments and these probably represent other P-glycoprotein gene family members [87]. Similarly, lowering the stringency of hybridization in Southern blot analysis with the human mdrl probe allowed the detection of cross-hybridizing DNA fragments [105]. Thus, Southern blot analysis of cell lines of diverse origin with a variety of P-glycoprotein cDNA or genomic DNA probes consistently indicated the presence of a family of P-glycoprotein genes.

III-C.4. The P-glycoprotein amplicon In the study of genes whose overexpression

correlates with development of multidrug resistance, six classes of cDNA clone, corresponding to six genes that are amplified and overexpressed in the multidrug-resistant CHO cell line CHRC5, were isolated. The class 2 cDNAs were shown to code for P-glycoprotein, but the functional significance of the other five genes for the multidrug-resistance phenotype is unknown (see subsection III-A.2). Studies of other biological systems indicate that gene amplification is often accompanied by co-amplification of large stretches of flanking DNA encompassing several other genes. Within the amplified unit or amplicon, there may be a gradient of amplification, with the highest level of amplification often being seen near the midpoint of the amplicon [106]. To investigate whether the entire array of genes found to be amplified in the CHRC5 cell line resided within one amplicon, large (100-1000 kb) DNA fragments obtained with infrequently cutting restriction enzymes and separated by pulse-field gradient gel electrophoresis were analyzed in Southern blots using the various classes of cDNA as probes. It was shown that class 1, 2 and 3 cDNAs hybridized to one DNA fragment and class 4 and 5 cDNAs to another. These two large DNA fragments in turn appeared to be adjacent, so these five genes are probably linked within one large segment of amplified DNA within the CHRC5 genome. This amplified unit was estimated to be at least 1100 kb in length. The class 6 gene was subsequently shown to be linked

114

to the class 4 and 5 genes and thus it must also be located within the same amplified DNA segment [84,90].

In situ hybridization of class 2, 4 and 5 gene probes to chromosome spreads of CHRC5 cells showed that amplified copies of all three genes could be localized to both HSRs that are characteristic of the CHRC5 karyotype but are absent in the parental cell line AuxB1. Moreover, the native (non-amplified) copy of all three genes was found to be situated in the same region on chromosome lq. These results taken together with the findings from pulse-field gradient gel electrophoresis indicate that the six gene classes are closely juxtaposed in the wild-type hamster genome and become amplified as one amplicon in CHRC5 cells [92].

In Southern blot analysis of CHRC5 genomic DNA, two levels of amplification are observed for DNA fragment hybridizing to the class 1-class 5 cDNAs [84]. In particular, the class 2 (P-glycoprotein) DNA fragments are subdivided into two groups that are amplified to different levels, suggesting that they may be two P-glycoprotein genes, both of which are recognized by the class 2 cDNA probes. Analysis of three other independently derived multidrug-resistant hamster cell lines revealed additional patterns of differential amplification of DNA fragments corresponding to the five genes. The DNA fragments hybridizing to class 2 probes show a more complex pattern of amplification levels that is consistent with the existence of a family of three P-glycoprotein genes (also see subsection III-C.3). A tentative map of the relative positions of these five genes within the amplicon was drawn, based on the results of pulse-field gradient gel analysis of the CHRC5 genome and on the patterns of differential amplification of these genes in four multidrug-resistant hamster cell lines. Genes that were amplified to the same level in different multidrug-resistant cell lines were assigned to neighbouring positions. According to this map (Fig. 1), the P-glycoprotein gene family of three members is flanked by genes 1, 3, 4 and 5 [91]. The position of the class 6 gene has not been determined, except that it is most closely linked to gene classes 4 and 5 [90].

Among the four independently derived multidrug-resistant hamster cell lines that were studied,

one or more of the P-glycoprotein (class 2) genes were consistently found to be amplified, while gene classes 1, 3, 4 and 5 were only amplified in some of the cell lines (Fig. 1). This agrees with other experimental evidence presented throughout this section (see summary to Section III) that overproduction of P-glycoprotein is necessary and sufficient for multidrug resistance. It seems likely that P-glycoprotein gene amplification is selected for during exposure to drugs such as colchicine, resulting in multidrug resistance, and the flanking genes are co-amplified to a variable extent due to their location contiguous to the P-glycoprotein genes. The contribution of the flanking genes to the multidrug-resistance phenotype is not known. Since functionally related genes may be clustered in the genome, it is possible that at least some of the co-amplified genes may contribute to multidrug resistance. Conceptually, variation in the boundaries of the amplicon and in its topography (amplification levels of the genes within its boundaries) can occur not only among independently derived multidrug-resistant cell lines but also within a series of cell lines selected for increasing degrees of multidrug resistance. Such variation may contribute to the complexity of the multidrug-resistance phenotype.

The products encoded by the co-amplified genes are not known, except for the class 4 gene. This gene was found to code for a small, phosphorylated cytosolic protein known variously as sorcin/V19 or CP22. Sorcin is overproduced in many, but not all, hamster, human and mouse multidrug-resistant cell lines selected for resistance to colchicine, vinca alkaloids, doxorubucin or actinomycin D [67,107]. Sequence analysis of hamster sorcin indicates that it has substantial homology with calcium-dependent proteinases termed calpains and contains structural units typical of calcium-binding sites. It is tempting to speculate that the partial reversal of multidrug resistance by some calcium-channel blockers and calmodulin inhibitors (see subsection II-B.2) may be linked to the overproduction of sorcin. Further studies to elucidate the role of sorcin and the currently unknown products of the other genes of the amplicon in modulation of the multidrug-resistance phenotype may increase our understanding, not only of the multidrug-resistance pheno-

type itself, but also of gene amplification in mammalian cells in general.

III-D. DNA-mediated transfer of multidrug resistance

Several independent studies have demonstrated that transfection with high-molecular-weight DNA from multidrug-resistant cells results in expression of the multidrug-resistance phenotype in previously drug-sensitive recipient cells [56,90,108-110]. In these studies, the selection protocol used to identify multidrug-resistant transfectants was carefully chosen to minimize the appearance of spontaneous multidrug-resistant variants among recipient cells. Appropriate experimental controls were utilized to show that acquisition of multidrug resistance was the direct result of transfection of DNA sequences fro/n multidrug-resistant cells. With the availability of various P-glycoprotein gene probes and monoclonal antibodies against P-glycoprotein, it was possible to demonstrate that DNA-mediated transfer of multidrug resistance was invariably associated with transfer and overexpression of P-glycoprotein sequences. This argues strongly that P-glycoprotein overexpression is necessary for multidrug resistance [90,108,109].

In one such study, drug-sensitive mouse LTA cells were transfected with DNA from the multidrug-resistant CHO cell line CHRC5 [90]. Inde- pendently derived, multidrug-resistant transformant clones were obtained after a stringent, three-step selection with colchicine designed to minimize the outgrowth of spontaneous, mouse multidrug-resistant variants. Southern blot analysis of the multidrug-resistant transfectants demonstrated the presence of amplified hamster P-glycoprotein sequences over a background of unampli- fled, endogenous mouse P-glycoprotein sequences. The transfected and amplified donor P-glycoprotein sequences were expressed, since all multidrug- resistant transfectants were found to overexpress P-glycoprotein on immunoblot analysis. By using appropriate monoclonal antibodies that dis- criminated between P-glycoprotein of hamster and mouse origin, it was ascertained that the overexpressed P-glycoprotein was of hamster (donor) origin. A further observation in the multidrug-resistant mouse transformants was that only a subset of hamster P-glycoprotein DNA sequences was

115

transfected. In four independently derived transfectants, the same subset of P-glycoprotein sequences was transferred. Preliminary analysis of transfectant DNA using probes specific for various P-glycoprotein gene family members indicated that the transfected hamster P-glycoprotein sequences corresponded to pgpl (Deuchars, K., unpublished results). Thus, it appears that overexpression of a single member of the P-glycoprotein multigene family results in the entire pleiotropic phenotype of cross-resistance to multiple drugs and collateral sensitivity to membrane-active agents. The basis for preferential involvement of one gene family member, pgpl, in transfection of multidrug resistance is not clear at present, but may reflect structural and functional differences among the gene family members.

The P-glycoprotein gene family was previously found to be closely linked to five other genes in the CHRC5 genome and the entire group of contiguous genes is amplified and overexpressed in this multidrug-resistant cell line (see subsection III-C.4). Analysis of four independent multidrug- resistant mouse transfectants showed that, in all cases, the five flanking genes were not co-transfected with the P-glycoprotein gene. Taken together, the characteristics of the multidrug-resistant mouse transfectants are completely consistent with the view that overexpression of P-glycoprotein is necessary and sufficient for multidrug resistance [90].

The successful transfer of the multidrug-resistance phenotype by transfection of P-glycoprotein DNA sequences across species in several studies [90,108,109] illustrates the high degree of structural and functional conservation of P-glycoprotein, at least among mammalian cells. In addition, the ability to transfer multidrug resistance through DNA transfection across species was used to ad- vantage by one group of investigators to clone gene(s) associated with multidrug resistance [110]. In this study, primary and secondary multidrug- resistant DNA transformants were obtained by transfecting drug-sensitive mouse LTK cells with high-molecular-weight DNA from the human multidrug-resistant cell line K562/ADM. DNA sequences of donor (human) origin that were consistently present in independently isolated transformants were identified in Southern blots using a

116

probe for human-specific Alu sequences. Since the amount of donor DNA remaining in secondary DNA transformants was small, donor DNA sequences that were invariably present in several secondary transformants probably corresponded to gene(s) necessary for multidrug resistance. One of the fragments identified in this manner was cloned by screening a genomic DNA library of a secondary transformant with a probe for human Alu sequences. This 2.6 kb DNA fragment (?~KA 2.6) was shown to recognize a 4.5 kb mRNA which was overexpressed in K562/ADM and 2780 AD cells, two human multidrug-resistant cell lines known to overexpress P-glycoprotein. Thus, this cloned human genomic DNA fragment probably represents a portion of a P-glycoprotein gene (Ta- ble IV).

The isolation of a full-length mouse cDNA clone encoding P-glycoprotein (?~DRll) and transfection of this cDNA in an expression vector into drug-sensitive cells demonstrated directly that overexpression of a single P-glycoprotein gene is sufficient to produce the multidrug-resistance phenotype [94,102]. In the first study, where drug-sensitive hamster LR73 cells were used as recipient cells, it could be readily shown in a number of independent transfectants that acquisition of the multidrug-resistance phenotype was consistently associated with overexpression of the transfected P-glycoprotein gene, while endogenous P-glycoprotein sequences were not overexpressed [94]. The transfected P-glycoprotein cDNA clone was derived from a cDNA library of a drug-sensitive mouse pre-B cell line by screening with hamster mdr/P-glycoprotein probes (see subsection III- A.3). Thus, overexpression of a normal P-glycoprotein gene can confer the multidrug-resistance phenotype, that is, development of multidrug-resistance is not contingent upon a structural muta- tion of the P-glycoprotein gene. In the second study, ~DRl l was co-transfected with the neomycin-resistance gene and multidrug-resistant transfectants could be obtained by selection with the neomycin analogue, G418, which is not included in the multidrug-resistance phenotype. Thus, multidrug resistance can result directly from increased transcription of ~DRl l in the expression vector without the need for selection with drugs such as adriamycin or colchicine that are

typically involved in the multidrug-resistance phenotype [102].

The mouse P-glycoprotein cDNA clone )~DRll has been shown to correspond to pgp2 in the P-glycoprotein multigene family that was characterized in drug-sensitive hamster cells [96]. The transfection studies described above showed that overexpression of mouse pgp2 results in the pleiotropic multidrug-resistance phenotype. Recently, a complete P-glycoprotein cDNA clone was con- structed by ligation of three partial human mdrl(P-glycoprotein) cDNA clones that were isolated from a colchicine-selected, multidrug-resistant KB/HeLa subline. When transfected into drug-sensitive mouse or human cells in an expression vector, this reconstructed cDNA clone con- ferred the multidrug-resistance phenotype onto the recipient cells [111]. The mdrl gene was found to be the human homologue of hamster pgpl [96]. Thus it appears that transfection and overexpression of either pgpl or pgp2 can confer the multidrug-resistance phenotype onto otherwise drug- sensitive cells. In the study of multidrug-resistant transfectants obtained by transfer of genomic DNA from CHRC5 cells to mouse LTA cells (see above, this section), Southern blot analysis using gene-specific probes also indicated that transfection of a single gene family member, pgpl, was sufficient for multidrug resistance. The limited analysis of the multidrug-resistance phenotype displayed by the various transfectants did not allow definite conclusions regarding any differences in the multidrug-resistance phenotype that might be attributed to overexpression of different members of the P-glycoprotein gene family. Among independently derived multidrug-resistant transfectants that overexpressed the same P-glycoprotein gene, there was significant variation of the cross-resistance profile. This suggested that factors such as post-translational modification of P-glycoprotein or characteristics of the milieu in which P-glycoprotein was overexpressed might also contribute to the variability of the multidrug-resistance phenotype [111].

III-E. Summary

A combination of several lines of evidence now clearly indicates that overexpression of P-glyco-

protein is necessary and sufficient for the multidrug-resistance phenotype. (i) Attempts to isolate genes whose amplification or overexpression is consistently associated with multidrug resistance have invariably led to one or more of the P-glycoprotein genes; thus, overexpression of P-glycoprotein genes is the predominant genetic alteration in multidrug resistance. (ii) The use of P-glycoprotein gene probes in molecular genetic analysis of numerous independently obtained multidrug-resistant cell lines, together with the respective drug-sensitive parent and revertants to drug sensitivity, has shown that multidrug resistance is consistently associated with amplification and/or overexpression of P-glycoprotein genes. This agrees with previous phenotypic studies (subsection II-C) which demonstrated the consistent association between multidrug resistance and overexpression of P-glycoprotein at the protein level. (iii) In independent studies of DNA-mediated transfection of multidrug resistance, transfection and overexpression of a P-glycoprotein gene alone, free of contiguous or linked sequences is sufficient to confer multidrug resistance upon otherwise drug-sensitive cells. (iv) Sequence analysis of P-glycoprotein genes reveals structural features consistent with a membrane-associated, energy-dependent molecular 'pump', thus providing a model for P-glycoprotein function that agrees well with the finding of enhanced energy-dependent drug efflux in multidrug-resistant cells (see Section IV).

The genomic organization and control of expression of P-glycoprotein genes have been partly elucidated through the study of both drug-sensitive and multidrug-resistant cell lines. P-glycoprotein is encoded by a small family of genes that are linked in the genome; the gene family in turn is linked to contiguous genes within an amplicon [91]. The size of one P-glycoprotein gene has been estimated to be approx. 75 kb [87] and the amplicon, as delineated in the multidrug-resistant CHO cell line CHRC5, spans more than 1100 kb [84]. There is evidence for differential expression of genes within this large domain, both in drug-sensitive cells and in multidrug-resistant derivatives [91,96]. Furthermore, it has been shown that multiple transcripts may be generated from a single P-glycoprotein gene through the use of alternate polyadenylation sites and splice signals [96,103].

117

It has been demonstrated that overexpression of a single P-glycoprotein gene is sufficient to confer the multidrug-resistance phenotype. Cur- rent efforts are directed at understanding the genetic basis of the characteristic variability of this phenotype (see subsection II-E). A number of potential mechanisms have been considered. (i) Differential expression of P-glycoprotein gene family members may result in different cross-resistance profiles. It may be significant that differential expression of P-glycoprotein genes also occurs in cells and tissues not selected for multidrug resistance [96,103]. Thus the pattern of expression of P-glycoprotein genes may be important for both its physiological function in different tissues and its role in development of multidrug resistance. (ii) P-glycoprotein molecules with different structure and function may be generated from a single P-glycoprotein gene, especially by alternate splicing of transcripts. (iii) Genes that are adjacent to the P-glycoprotein gene family may modulate the multidrug-resistance phenotype, although DNA transfection studies have shown that their co-transfection with the P-glycoprotein gene is not required to confer multidrug resistance upon recipient cells. Although the function of these genes is at present unknown, it may be speculated that differential expression of these genes may contribute to the variability of the multidrug-resistance phenotype. The isolation of clones corresponding to various P-glycoprotein genes and to the contiguous genes in the amplicon, the identification of alternate splice sites and the development of DNA transfection assays to examine the function of isolated genes in multidrug resistance together should provide the armamentarium for further studies of the complex regulation of the multidrug-resistance phenotype.

IV. Structure of P-glycoprotein

P-glycoprotein has been characterized biochem- ically as an integral plasma membrane glycoprotein that spans the lipid bilayer. Studies of intact cells showed that the portion of P-glycoprotein exposed at the cell surface is reactive with the galactose oxidase-boro[3H]hydride technique for labelling surface carbohydrates and is also susceptible to proteolytic digestion under conditions

118

where only cell surface peptides are attacked [54,55]. Mapping of epitopes with monoclonal antibodies led to localization of the C-terminus of the molecule on the cytoplasmic side of the plasma membrane [58].

IV-A. Sequence analysis of P-glycoprotein - implications for its function

Further structural analysis of P-glyc0protein was accomplished through sequence analysis of cDNA clones from mouse, hamster and human cell lines, cDNA clones that encompass the entire coding region of a P-glycoprotein gene have been derived from a drug-sensitive mouse cell line [95] and from a highly drug-resistant human cell line [97]. Sequence analysis of these clones allowed the complete amino-acid sequence of the respective P-glycoprotein molecules to be deduced. There are 1276 amino acids in the mouse sequence and 1280 amino acids in the human sequence and the calculated molecular masses are 140 and 141 kDa, respectively. These values are in complete agreement with the estimated size of 140 kDa for the polypeptide portion of P-glycoprotein [112]. In each case, the P-glycoprotein molecule consists of a tandem duplication. The duplicated moiety may be considered to have a N-terminal portion and a C-terminal portion. The C-terminal portion of the two halves of the molecule are highly homologous

and each contains the consensus sequence for an ATP-binding site. While the corresponding N- terminal portions show less striking amino-acid sequence homology, a hydropathy plot of these two segments demonstrates a markedly similar arrangement of six possible transmembrane regions arranged in three pairs. Thus, the P-glycoprotein molecule appears to consist of a repeated arrangement of a transmembrane segment associated with an ATP-binding domain (Fig. 2).

Partial P-glycoprotein cDNA clones have been obtained from a drug-sensitive CHO cell line [89,96] and from a multidrug-resistant CHO cell line [84]. Sequence analysis of these overlapping clones led to the identification of two homologous hamster P-glycoprotein genes, pgpl and pgp2 (see subsection III-B.3). In each case, the sequence data are consistent with a tandemly duplicated molecule, in agreement with the findings for the mouse and human P-glycoprotein genes. The longest cDNA clone from the drug sensitive cell line (pL20) encompasses the C-terminal 655 amino acids and essentially constitutes the duplicated moiety of the P-glycoprotein molecule [96].

Comparison of the sequence of pL20 against the nucleic acid sequence database revealed striking homology at the level of the deduced amino- acid sequence with hlyB, a bacterial membrane protein present in hemolytic strains of E. coli which is required for export of a-hemolysin (a

r~ N N D ~ ND~ • ~ ~ P . g l y c o p r o t e i n

I ~ D¢~ ND~ • ~ ] H l y B

H i s P z/z ~ j

M a l K [ " ~ I

R b s A Jm r~ • ~ t

w h i t e l o c u s ~" • ~ ~ ~ ~n

i t 1 0 0 a m i n o a c i d s

Fig. 2. Relative location of transmembrane and nucleotide-binding domains of P-glycoprotein and various bacterial and Drosophila transport-associated proteins. The crossed boxes represent putative transmembrane domains of 21 amino acids in length which were identified as described in Ref. 89. The black and hatched boxes represent the highly conserved sequences of 24 and 32 antino acids (defined in Ref. 138) which are involved in nucleotide-binding. HlyB gene product is a bacterial membrane protein required for secretion of a-hemolysin from E. coli. The ATP-binding proteins HisP, MalK and RbsA are analogous members of bacterial multicomponent, substrate transport systems involved in the transport of histidine, maltose (and maltodextrin) and ribose, respectively. The white locus functions in pigment transport in Drosophila melanogaster. The left and right hand side of the bars

represent the amino- and carboxy-terminal of the protein, respectively.

protein of 107 kDa) to effect hemolysis [89]. The deduced amino-acid sequence in each case con- tained an extensive, hydrophobic segment at the N-terminus. Although the degree of amino-acid homology is not striking here, hydropathy plots of this region are remarkably similar and indicate the presence of six potential transmembrane segments arranged as three pairs. The hydrophobicity and hydrophobic moments of these putative transmembrane segments are within the range observed for channel-forming proteins. For the P-glycoprotein molecule, the orientation of the transmembrane segments within the plasma membrane is assigned so that the C-terminus of the molecule is on the cytoplasmic side of the plasma membrane, as established previously by monoclonal antibody studies. In this orientation, the three pairs of transmembrane segments are separated by relatively long stretches of amino acids on the cytoplasmic face of the plasma membrane, while only short stretches of amino acids are present on the cell surface. Examination of the C-terminal aspect of the two amino-acid sequences shows a high degree of sequence homology, with the regions of greatest homology including and extending beyond the consensus sequence for an ATP-binding site [89].

Alignment of the P-glycoprotein sequences derived from mouse, human and hamster cells and the hlyB sequence shows a highly conserved overall structure of an N-terminal transmembrane portion that has characteristics of a channel-forming domain and a C-terminal intracytoplasmic domain that includes a putative ATP-binding site. The P-glycoprotein molecule appears to have arisen as a tandem duplication of a hlyB-like molecule (Fig. 2). There is a remarkable degree of conservation of amino-acid sequence within the C-terminal end of the duplicated moiety, not only among P-glycoprotein molecules of different mammalian species but also between P-glycoprotein and the bacterial protein hlyB. The highest concentration of amino-acid identities is in the region that encompasses the putative ATP-binding fold [89,96].

Further insight into the functional significance of this highly conserved section of the P-giycoprotein molecule was obtained when it was found to be also highly homologous to a number of ATP-

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binding bacterial transport proteins - hisP, malK, oppD, pstB and rbsA (Fig. 2). These are all analogous members of multicomponent, high-affinity substrate-transport systems composed of a peri- plasmic substrate-binding protein, two hydrophobic, integral membrane proteins and the ATP- binding protein. These ATP-binding components of transport systems have been shown to share long stretches of amino acids within their sequences that extend much beyond the fairly short consensus sequences for ATP-binding. This finding indicates the presence of a shared functional unit in these transport proteins. Recently, this functional unit was identified through sequence homology in a number of other proteins of more diverse functions, including hlyB (already described above), ftsE (cell division in E. coli), nodI (nodulation in the nitrogen-fixing bacterium Rhizobium leguminosarum), uvrA (DNA repair in E. coli) and the white locus (pigment transport in Drosophila melanogaster) [113,114,138]. It was postulated that this functional unit was concerned with ATP-binding and coupling of energy production from ATP hydrolysis to biological processes that were commonly, but not exclusively transport processes [113]. Amino-acid sequence comparison between P-glycoprotein and the bacterial transport proteins (hisP, malK, oppD and pstB) clearly shows that the C-terminal portion of each half of the P-glycoprotein molecule contains this ATP- binding functional unit [89].

A rather surprising finding is that the degree of homology between P-glycoprotein (a mammalian protein) and the bacterial transport proteins is comparable to the degree of homology within the bacterial transport proteins themselves. In fact, hlyB is more homologous to P-glycoprotein than it is to hisP, malK, oppD and pstB [89]. The extent of sequence conservation through such a long evolutionary span points to a strong selection pressure due to functional requirements. It seems likely that this portion of P-glycoprotein is concerned with ATP-binding and coupling of energy production to transport.

IV-B. Homology of P-glycoprotein with other transport systems - evolutionary considerations

The striking homology between each half of the P-glycoprotein molecule and the bacterial mem-

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brane protein hlyB suggests that the P-glycoprotein gene may have originated with the tandem duplication of an ancestral hlyB-like gene. The significance of the duplication is not known, but it is interesting to note that one of the bacterial ATP-binding transport proteins, rbsA, which shares homology with hlyB and P-glycoprotein in the ATP-binding domain, also exists as a tandemly duplicated molecule (Fig. 2). Moreover, there is some evidence that at least some of the other bacterial transport proteins shown in Fig. 2 may function as homodimers or heterodimers [113]. The ancestral P-glycoprotein gene may then have evolved into a multigene family through additional occurrences of gene duplication. Com- parative sequence analysis of the duplicated moie- ties within the complete mouse P-glycoprotein sequence (corresponding to P-glycoprotein gene 2, or pgp2, see subsection III-B.3) and within the complete human P-glycoprotein sequence (corresponding to pgpl) demonstrate that there is a greater degree of sequence homology between corresponding halves of the two P-glycoprotein genes than between the two halves of each gene. Al- though this should be confirmed by examination of the complete sequence of P-glycoprotein gene family members isolated from one species, the available sequence data are consistent with the hypothesis that tandem duplication to produce the full-length P-glycoprotein molecule preceded the development of the P-glycoprotein multigene family. Sequence analysis of the 3'-half of different hamster P-glycoprotein genes (pgpl, 2 and 3) reveals that intron-exon organization is conserved among gene family members, indicating that this was already established prior to the development of the gene family (Ng, W. and Ling, V., unpublished data).

The isolation of P-glycoprotein DNA sequences, predominantly in the form of partial or complete cDNA clones, from cells of hamster, mouse and human origin [95,96,98,103] has allowed the comparative study of the P-glycoprotein gene family in different mammalian species. The existence of a P-glycoprotein gene family was first directly demonstrated in hamster cells [96]. The results of sequence analysis of P-glycoprotein cDNA clones derived from mouse and human cells is completely consistent with the parallel

existence of a gene family in these species (see subsection III-B.3) The gene family structure appears to be conserved across species, although the pattern of expression of P-glycoprotein genes has been found to vary among different cells and tissues. Thus, it is hypothesized that divergence of the gene family members predated evolution into the mammalian species. A corollary of this hypothesis is that, since the sequence homology between hamster pgpl and pgp2 is greater than expected, gene conversion may have occurred among gene family members of one species [96]. The extent of occurrence of gene conversion among P-glycoprotein gene family members of various species and its functional implications await sequence analysis of additional P-glycoprotein genes and investigation into structure-function relation- ships of the P-glycoprotein molecule.

V. Model for P-glycoprotein function

The information obtained from amino-acid sequence analysis of P-glycoprotein from mouse, human and hamster cells may be organized into a model for P-glycoprotein (Fig. 3): P-glycoprotein forms a channel in the plasma membrane and transports drugs out of cells using energy derived from ATP hydrolysis. The number of P-glycoprotein molecules required to form one channel is not known. In one version of this model (Fig. 3), P-glycoprotein binds drugs directly and then re- moves them from the cell. There are two pertinent considerations here: (i) drug binding to P-glycoprotein must be reversible, since drug molecules have to be released at the cell surface; (ii) since transfection of a P-glycoprotein cDNA clone into drug-sensitive cells results in cross-resistance to structurally unrelated drugs (see subsection Ill-D), the P-glycoprotein molecule must have binding- sites for a diverse group of drugs, probably within its hydrophobic domain [89].

There is experimental evidence to support a drug-binding function for P-glycoprotein. Mem- brane vesicles from both multidrug-resistant Chinese hamster lung cell lines and multidrug-resistant human carcinoma cell lines have been shown to overexpress a protein of 150-180 kDa which has specific binding-sites for vinblastine, as

Fig. 3. Model of P-glycoprotein as a channel-forming energy-dependent export pump, based on sequence analysis. It is postulated that the twelve alpha-helical transmembrane domains (here drawn as cylinders embedded in the lipid bilayer) form a pore. Although a single P-glycoprotein molecule is shown, it is possible that several molecules may be required to form the channel. The nucleotide-binding domains (ATP) have been localized to the cytoplasmic side using monoclonal antibodies. The site(s) of N-linked glycosylation (chain of circles) is presumed to lie on the short extraceUular peptide fragment located at the amino-terminal end of the protein between the first and second transmembrane domains (Refs. 95, 97). Drugs (triangles and hexagons), which enter the cell by diffusion, are vectorially exported from the cell either directly through the pore or indirectly following binding to a carrier molecule

which may be a peptide or protein.

shown by photoaffinity-labelling with radioactive vinblastine analogs [115,116]. This labelling is inhibited by two of the drugs which are cross-resistant with vinblastine in multidrug-resistant cells (vincristine, and to a lesser extent, daunorubicin) [116]. This suggests that these drugs may be com- peting for the same binding-site or binding to closely adjacent sites. The identity of the 150-180 kDa vinblastine binding protein in multidrug-resistant human cells with P-glycoprotein was shown by immunoprecipitation with a monoclonal antibody against P-glycoprotein [43]. It has not been established whether P-glycoprotein also binds other drugs involved in the multidrug-resistance

121

phenotype, such as colchicine and actinomycin D which were shown not to compete for the vinblastine binding-site in membrane vesicles in the experiments mentioned above [116].

Further evidence that drug-binding is involved in the function of P-glycoprotein in multidrug resistance came from studies of various agents that p a r t y or completely reverse multidrug resistance (see subsection II-B.2). Many, but not all, of these agents inhibit photoaffinity labelling of P- g lycoprote in by the vinblast ine analogue [125I]NASV [140]. Thus, reversal of multidrug resistance may occur through inhibition of drug- binding by P-glycoprotein. Two observations suggests that there may be multiple drug-binding domains in P-glycoprotein: (i) inhibition of vinblastine-analog binding to P-glycoprotein was not commensurate with ability to reverse multidrug resistance for several compounds, such as trifluoperazine and chloroquin; (ii) drugs, such as colchicine and actinomycin D, that are involved in the multidrug-resistance phenotype did not compete for the vinblastine-binding site in membrane vesicles of multidrug-resistant cells [116,140].

Recently, a photoact ive calcium channel blocker, [3H]azidopine, was shown to bind to P- glycoprotein [146,147]. This binding was inhibited by other calcium channel blockers and by drugs such as vinblastine, actinomycin D, colchicine, adriamycin and taxol when these drugs were present in excess. The availability of photoactive analogs of vinblastine, such as [125I]NASV, and of [3H]azidopine as probes for drug-binding domain(s) in the P-glycoprotein molecule should prove useful for studying the structure and function of P-glycoprotein.

In the second version of the model for P-glycoprotein function, a drug-binding protein is trans- ported out of cells by the P-glycoprotein 'pump' analogous to the export of hemolysin by HlyB in E. coli (Fig. 3). Drugs may bind irreversibly to this protein and the entire drug-protein complex is removed from the cell. The hypothetical drug- binding protein may be a normally expressed cellular constituent, but it must be produced in sufficient quantities, as it is continuously exported [89].

The presence of ATP-binding sites on the P- glycoprotein molecule was first deduced from sequence analysis [89,95,97]. Nucleotide-binding by

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P-glycoprotein was directly demonstrated by labelling membrane vesicles with the photoaffinity reagent 8-azido-[a-32p]ATP and immunoprecipitation with a monoclonal antibody against P-glycoprotein [117]. Recently, P-glycoprotein was purified from membrane extracts of a multidrug- resistant human leukaemia cell line (K562/ADM) by means of immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein. The purified P-glycoprotein was shown to have ATPase activity. The ATPase activity of purified P-glycoprotein was lower than that of other membrane pumps such as Na+/K+-ATPase. It remains to be determined whether ATP hydrolysis by P-glycoprotein is coupled to drug efflux, as predicted by the current model of P-glycoprotein function [148].

This model of P-glycoprotein as an ATP-dependent efflux channel agrees well with studies of drug transport and accumulation which show that for vinca alkaloids and anthracyclines, there is an ATP-dependent drug efflux system (see subsection II-B). However, this model must represent a sim- plified concept of the mode of function of P-glycoprotein that will require modifications according to ~tdditional experimental findings. For example, different P-glycoprotein gene family members may code for slightly different P-glycoprotein molecules and these may participate in the formation of a more complex functional unit. In addition, the exaggerated presence of a complex molecule such as P-glycoprotein within the plasma membrane in multidrug-resistant cells may be expected to have multiple effects on membrane structure and function, including membrane permeability and response to membrane-active agents such as ionophores, local anaesthetics and non- ionic detergents. In particular, resistance to colchicine in multidrug-resistant CHO cells has been well characterized and shows features that are most consistent with alterations in an energy-dependent permeability barrier. Colchicine binds with great affinity to its intracellular target, tubulin, and it seems unlikely that a drug efflux mechanism such as that depicted in Fig. 3 can compete with tubulin for intracellular colchicine. Similarly, the remarkable degree of cross-resistance shown by several multidrug-resistant cell lines to the ionophore Gramicidin D indicates that P-glyco-

protein must mediate other aspects of membrane function in addition to efflux of specific molecules.

A recent study of plasma membrane ultrastruc- ture using freeze-fracture analysis revealed increases in the density of protoplasmic face intramembrane particles (IMP) in multidrug-resistant CHO and human leukaemic cells. The increase in IMP correlated with increase in P-glycoprotein content and multidrug resistance. These findings support the hypothesis that overexpression of P-glycoprotein leads to global changes in plasma membrane architecture that in turn results in a variety of membrane-related alterations which are not easily explained by the efflux pump model of P-glycoprotein function [118].

Various post-translational modifications of the P-glycoprotein molecule such as glycosylation and phosphorylation should be considered as possible mechanisms for modulation of its function. P-glycoprotein contains a fairly large carbohydrate component that accounts for approx. 20% of its relative molecular mass. A phytohaemagglutinin- resistant derivative of the multidrug-resistant CHO cell line CHRC5 contains P-glycoprotein of lower molecular mass (approx. 140 kDa) due to a substantial loss of its carbohydrate content. This alteration has no apparent effect on multidrug resistance and collateral sensitivity [112]. Lack of influence of the carbohydrate portion of P-glycoprotein on drug resistance was also shown by growing multidrug-resistant human leukaemic cells in tunicamycin to block glycosylation of P-glycoprotein and by pronase treatment of multidrug-resistant cells to remove cell surface glycopeptides. Neither of these treatments appeared to alter the drug resistance characteristics of the cells [119]. Variation in N-linked oligosaccharide was demonstrated among heterogeneous forms of P-glycoprotein that are overexpressed by different multidrug-resistant mouse J774.2 cell lines (see subsection II-C). These multidrug-resistant cell lines exhibit distinct cross-resistant profiles, but it has not been determined whether the difference in carbohydrate component of P-glycoprotein is the basis of the difference in response to drugs [141].

P-glycoprotein has been shown to be phosphorylated in vivo (Refs. 120, 121; Georges, E. and Ling, V., unpublished data) and in vitro

[29,120]. In addition, pulse chase experiments indicated that P180 (P-glycoprotein) was rapidly phosphorylated and dephosphorylated (within minutes) in vivo [120]. Quantitative evaluation of the changes in phosphorylation of P-glycoprotein demonstrated in this study is difficult because it is not clear if the total amount of 32p label in membrane proteins remained constant during the experiments. An unequivocal demonstration of rapid phosphorylation and dephosphorylation of P-glycoprotein in vivo would support the hypothesis that phosphorylation modulates the dynamics of P-glycoprotein function.

Alterations in the state of phosphorylation of P-glycoprotein appears to be involved in the mechanism of action of several compounds that enhance drug accumulation in multidrug-resistant cells, resulting in circumvention of drug resistance (see subsection II-B.2). Thus, the metabolic inhibi- tor N-ethylmaleimide (NEM) increased adriamycin accumulation in multidrug-resistant Chinese hamster lung cells but not in the drug-sensitive parent. Treatment of 32p-labelled cells of the resistant subline with NEM at similar concentrations for 20 min resulted in an apparent increase in phosphorylation of P-glycoprotein. 'Superphos- phorylation' of P-glycoprotein was also demonstrated upon treatment of 32p-labelled, multidrug- resistant Chinese hamster lung cells for 20 min with trifluoperazine or verapamil, at concentrations that effectively increased drug accumulation in these cells [120]. As in the case of the pulse chase experiments described above, it is not clear if the total amount of 32p label in membrane proteins was unchanged after incubation with NEM, trifluoperazine or verapamil, so that other explanations for the apparent 'superphosphorylation' of P-glycoprotein are possible.

The finding of increased phosphorylation of P-glycoprotein by trifluoperazine and verapamil was confirmed recently in multidrug-resistant human leukaemic cells (K562/ADM), where it was shown that treatment with these agents did not change the total amount of 32p label in membrane proteins [121]. In the same study, it was also shown that treatment of multidrug-resistant K562/ADM cells with a phorbol diester, 4/3- phorbol 12/3-myristate 13a-acetate (PMA), or with 1-oleoyl-2-acetylglycerol (OAG), resulted in

123

increased phosphorylation of P-glycoprotein. This suggested that P-glycoprotein in these cells might be a substrate for activated protein kinase C. Analysis of tryptic digests of 32P-labelled P-glycoprotein revealed differences in the sites of phosphorylation of P-glycoprotein from untreated K562/ADM cells and verapamil or trifluopera- zinc-treated K562/ADM cells on one hand and PMA-treated K562/ADM cells on the other hand. Thus, the phosphorylation of P-glycoprotein is likely to be regulated by more than one mechanism, each controlling phosphorylation at different sites. While 'superphosphorylation' of P-glycoprotein in K562/ADM cells by verapamil or trifluoperazine was thought to be involved in circumvention of multidrug resistance by these agents, the effect of PMA-induced increase in phosphorylation of P-glycoprotein on multidrug resistance was not determined in this study [121].

Changes in phosphorylation of cellular proteins were found to be associated with resistance to vincristine and adriamycin in a breast carcinoma cell line (MCFT) [149]. Cells were exposed to a phorbol ester, phorbol 12,13-dibutyrate (P(BtO)2), which stimulates protein kinase C activity. A general increase in protein phosphorylation as well as a specific increase in phosphorylation of a 20 kDa particulate protein was demonstrated following treatment with P(BtO)2. This was associated with reduced accumulation of both vincristine and adriamycin and increased resistance to these drugs in a colony-forming assay, when compared with cells not treated with P(BtO)2. The induction of drug resistance by treatment with phorbol ester was more pronounced in wild-type MCF7 cells than in the multidrug-resistant derivative MCF7 Dox R. The MCF7 Dox R cell line has been shown to overexpress P-glycoprotein [126], but the effect of P(BtO)2 on phosphorylation of P-glycoprotein was not studied. The role of the 20 kDa particulate protein in multidrug resistance is unclear, although previous studies have shown increased phosphorylation of this protein in multidrug-resistant human small cell lung carcinoma and human breast carcinoma cell lines when compared to the drug-sensitive parent cell lines.

In the same study, verapamil and trifluopera- zinc were found to partly reverse multidrug resistance in the MCF7 Dox R cell line. Changes in

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protein phosphorylation also appeared to be involved in the reversal of multidrug resistance by these compounds, since they reduced the overall level of phosphorylation of cellular proteins and also decreased the phosphorylation of the 20 kDa particulate protein in MCF7 Dox R cells. In addit ion, verapamil and tr i f luoperazine antagonized the effect of P(BtO)2 in increasing phosphorylation and inducing resistance to vincristine and adriamycin. It was not determined whether verapamil affected cellular response to P(BtO)2 through inhibiting protein kinase C activation by P(BtO)2 or through other mechanisms. The effect of verapamil and trifluoperazine on level of phosphorylation of P-glycoprotein was not measured [149].

The relationship between ATP-binding by P-glycoprotein and its phosphorylation is not known, although it is tempting to suggest that these reactions are linked, especially since metabolic inhibitors such as NEM affect both ATP levels and level of phosphorylation of P-glycoprotein. On the other hand, verapamil and trifluoperazine are not known to alter ATP levels but are able to cause 'superphosphorylation' of P-glycoprotein, perhaps by modulating the activity of kinases or phosphatases or by inducing conforma- tional changes in the P-glycoprotein molecule to expose sites for phosphorylation [121].

In summary, a model of P-glycoprotein as an ATP-binding drug efflux pump is consistent with its structural (amino-acid sequence) features and supported by experimental findings on drug transport and accumulation in multidrug-resistant cells. This model can provide a basis for further understanding of the pleiotropic multidrug-resistance phenotype, especially in the following areas: (i) mechanisms through which P-glycoprotein also mediates alterations in diverse membrane properties such as drug permeability, sensitivity to membrane-active agents, fluidity and resistance to disruption; (ii) dynamic aspects of P-glycoprotein function and how this relates to its state of phosphorylation. Metabolic inhibitors and agents such as verapamil that circumvent multidrug resistance appear to interfere with P-glycoprotein at this level, since their effects are rapid and reversible and they also alter the state of phosphorylation of P-glycoprotein.

VI. Concluding remarks

Multidrug resistance has been studied exten- sively in mammalian cells. In different multidrug- resistant cells, the pattern of cross-resistance to structurally and functionally unrelated drugs is characteristically variable and cannot be predicted a priori. The basis of multidrug resistance is reduced drug accumulation. Kinetic studies have suggested that complex abnormalities in drug transport occur in multidrug-resistant cells to result in reduced accumulation of a wide variety of drugs. In particular, there is evidence that both drug efflux and drug permeability are altered in multidrug-resistant cells. Although the phenome- non of multidrug resistance is well documented, phenotypic studies have not yet revealed its underlying mechanism, one of the major obstacles in these studies being the complex nature of this phenotype.

The most consistent biochemical change in multidrug-resistant cells is overexpression of P- glycoprotein in the plasma membrane. Genetic studies have clearly established that overexpression of P-glycoprotein is the single genetic alteration required for the multidrug resistance phenotype. Genetic studies have also demonstrated the presence of a family of P-glycoprotein genes which in turn may be closely linked in expression to contiguous genes in an amplicon. Current investigations are directed at two fundamental questions: (i) the mechanism through which P-glycoprotein overexpression results in the pleiotropic multidrug-resistance phenotype; (ii) the basis of the variability of the phenotype and how this relates to the differential expression of P-glycoprotein gene family members and of the other genes of the P-glycoprotein amplicon. The first question has been partly answered with the dis- covery of sequence homology between P-glycopro- rein and a group of bacterial transport proteins. An ATP-dependent drug-efflux pump model has been proposed for P-glycoprotein on the basis of sequence analysis and comparison with the bacterial transport proteins. The model accounts for part of the cross-resistance profile of multidrug-resistant cells but it has not explained how P-glycoprotein also mediates other features of this phenotype, such as collateral sensitivity, altered

growth characteristics, etc. (see Section II). Since cloned P-glycoprotein genes that correspond to different members of the multigene family are now available, an obvious approach to answer these two questions is to relate structure and function of the P-glycoprotein molecule through in vitro mutagenesis of the P-glycoprotein genes. Findings from such molecular genetic studies will have to be correlated with the plethora of changes that have been documented in multidrug-resistant cells, to arrive at the molecular mechanism of this pleiotropic phenotype.

There is now evidence that P-glycoprotein is expressed in normal cells that have not been selected for multidrug resistance, possibly in a tissue-specific manner. The relationship between P-glycoprotein expression in multidrug-resistant cells and that in normal ceils is not clear at present. The finding that different P-glycoprotein gene family members are simultaneously expressed in drug-sensitive cells as well as in multidrug-resistant cells is of particular interest. It may be speculated that P-glycoprotein has a physiologic function that depends on the balanced expression of several P-glycoprotein genes. Unbal- anced expression of P-glycoprotein genes may result in an aberrant function, that is, multidrug resistance. A corollary to this hypothesis is that analysis of individual P-glycoprotein gene family members will be necessary in the study of P-glycoprotein expression in both drug-sensitive and resistant cells.

The central role of P-glycoprotein overexpression in multidrug resistance has been established. However, in those multidrug-resistant cell lines that were selected by continuous growth in cytotoxic drugs for prolonged periods, there may be additional, independent genetic alterations. Such alterations may include changes in DNA topoisomerase activity and in glutathione metabolism. Although such complex multidrug-resistant variants may be viewed as useful models of drug resistance in vivo, a fundamental understanding of the role of each genetic alteration in multidrug resistance can only be obtained by analysis of genetically well-characterized systems that allow correlation of a particular biochemical change with the development of multidrug resistance.

Although overexpression of P-glycoprotein may

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not be the only mechanism for multidrug resistance, intensive study of its role in multidrug resistance in vitro and in vivo is clearly an important approach towards the understanding of drug resistance in mammalian cells, for a number of reasons. The P-glycoprotein-mediated multidrug-resistance phenotype is complex and yet it is due to the altered expression of one specific protein family. It is highly conserved across mammalian species so that rodent multidrug-resistant cells may be used as models of the human coun- terpart. The phenotype is subject to modulation by a variety of compounds. Gene probes and transfection assays are available for molecular genetic analysis. Thus, the study of P-glycoprotein-mediated multidrug resistance promises to bring new insight into membrane biology as well as to provide a rational basis for improvement in clinical chemotherapy.

Acknowledgements

The studies in the authors' laboratory were supported by the National Cancer Institute of Canada and by Public Health Service grant CA37130 from the National Institutes of Health, P.F.J. is a Fellow of the Medical Research Council of Canada. We are grateful to a number of workers, quoted in the text, for providing us with unpublished information. We thank our colleagues for helpful discussions, and especially J. Endicott for critical reading of the manuscript.

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